Protease resistant flint analogs

ABSTRACT

The invention relates to FLINT analogs that are to proteolysis in vivo and in vitro at amino acid position 218 of mature FLINT, clinical and therapeutic uses thereof, and pharmaceutical formulations comprising said analogs.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application Nos.60/126,839, filed Mar. 30, 1999; 60/140,073, filed Jun. 21, 1999;60/147,071, filed Aug. 4, 1999; 60/160,524, filed Oct. 20, 1999;60/160,669, filed Oct. 21, 1999; 60/172,744, filed Dec. 20, 1999; andNo. 60/178,184, filed Jan. 26, 2000.

BACKGROUND OF THE INVENTION

A number of tumor necrosis factor receptor proteins (“TNFR proteins”)have been isolated in recent years, having many potent biologicaleffects. Aberrant activity of these proteins has been implicated in anumber of disease states.

One such TNFR homologue, referred to herein as “FAS Ligand InhibitoryProtein” or “FLINT”, binds FAS Ligand (FAS L) thereby preventing theinteraction of FAS L with FAS (See U.S. Provisional Applications Ser.Nos. 60/112,577, 60/112,933, and 60/113,407, filed Dec. 17, 18 and 22,1998, respectively, the entire teachings of which are incorporatedherein by reference).

Increased activation of the FAS-FAS Ligand signal transduction pathwayis implicated in a number of pathological conditions, including runawayapoptosis (Kondo et al., Nature Medicine 3(4):409-413 (1997); Galle etal., J. Exp. Med. 182:1223-1230 (1995)), and inflammatory diseaseresulting from neutrophil activation (Miwa et al.,. Nature Medicine4:1287 (1998)).

“Runaway apoptosis” is a level of apoptosis greater than normal, orapoptosis occurring at an inappropriate time. Pathological conditionscaused by runaway apoptosis include, for example, organ failure in theliver, kidneys and pancreas. Inflammatory diseases associated withexcessive neutrophil activation include sepsis, ARDS, SIRS and MODS.

Compounds such as FLINT, which inhibit the binding of FAS to FASL, andLIGHT to LTβR and/or TR2/HVEM receptors, can be used to treat or preventdiseases or conditions that may be associated with these bindinginteractions. The therapeutic utility of FLINT could be enhanced byFLINT analogs that exhibit modified pharmacological properties (e.g.,enhanced potency, and/or longer in vivo half-lives, and/or greateraffinity for FASL), modified pharmaceutical properties (e.g., decreasedaggregation and surface adsorption, increased solubility and ease offormulation) and/or modified physical properties such as susceptibilityto proteolysis.

SUMMARY OF THE INVENTION

The FLINT polypeptide undergoes proteolysis in vivo to produce at leasttwo major peptide fragments. One of the fragments consists of residues 1through 218 of SEQ ID NO:1 (alternatively residues 1 through 247 of SEQID NO.:3), termed herein “FLINT metabolite;” the other consists ofresidues 219 through 271 of SEQ ID NO:1 (alternatively residues 248through 300 of SEQ ID NO:3). Cleavage at the 218 position in vitro canbe achieved when native FLINT (SEQ ID NO:3), or mature FLINT (SEQ IDNO:1), is treated with a trypsin-like enzyme, for example, thrombin,trypsin or other serine protease. Thus it is likely that a serineprotease is responsible for the in vivo proteolysis of FLINT. Productionof FLINT metabolite is disclosed in co-pending U.S. patent applicationSer. No. 09/936,024, herein incorporated by reference.

In vitro studies suggest that FLINT metabolite binds FasL with anapparent lower affinity than FLINT. Therefore, the pharmaceuticalutility of FLINT could be enhanced by an analog that is resistant toproteolysis at or near the 218 position. The invention disclosed hereinprovides such analogs.

In one embodiment, the invention relates to a FLINT analog that isresistant to proteolysis between positions 218 and 219 of SEQ ID NO:1,and/or between positions 247 and 248 of SEQ ID NO:3 in vivo and/or invitro.

In another embodiment, the invention relates to a FLINT analog that issubstantially resistant to proteolysis between positions 218 and 219 ofSEQ ID NO:1, and/or between positions 247 and 248 of SEQ ID NO:3 in vivoand/or in vitro.

In another embodiment, the invention relates to a FLINT C) analog thatis resistant to proteolysis by a trypsin-like protease between positions218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQID NO: 3 in vivo and/or in vitro.

In another embodiment, the invention relates to a FLINT analog that isresistant to proteolysis by a serine protease, for example, trypsin,thrombin, or chymotrypsin between positions 218 and 219 of SEQ ID NO:1,and/or between positions 247 and 248 of SEQ ID NO:3, in vivo and/or invitro.

In another embodiment, the invention relates to a FLINT analog that isresistant to proteolysis by a trypsin-like protease between positions218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQID NO:3, said analog comprising a polypeptide that is at least about 80%identical; alternatively at least about 90% identical; alternatively atleast about 95% identical; alternatively at least 96% identical;alternatively at least 97% identical; alternatively at least 98%identical; alternatively still, at least 99% identical with SEQ ID NO:1and/or SEQ ID NO:3.

In another embodiment, the invention relates to a FLINT analog that isresistant to proteolysis by a trypsin-like protease between positions218 and 219 of SEQ ID NO:1, and/or between positions 247 and 248 of SEQID NO:3, said analog comprising a polypeptide that is at least about 10%identical; alternatively at least 20% identical; alternatively at least30% identical; alternatively at least 40% identical; alternatively atleast 50% identical; alternatively at least 60% identical; alternativelyat least 70% identical, alternatively at least 80% identical,alternatively still, at least 90% identical with residues 214 through222 of SEQ ID NO:1 and/or residues 243 through 251 of SEQ ID NO:3.

In another embodiment, the invention relates to a FLINT analogcomprising one or more amino acid substitutions) deletion(s), oraddition(s) in the region comprising amino acids 214-222 of SEQ ID NO:1and/or amino acids 243-251 of SEQ ID NO:3.

In another embodiment, the invention relates to a FLINT analogcomprising one or more amino acid substitutions), deletions, oraddition(s) in the region comprising amino acids 215-218 of SEQ ID NO:1and/or amino acids 243-251 of SEQ ID NO:3.

In another embodiment, the invention relates to a FLINT analogcomprising one or more amino acid substitution(s) in the region 214-222of SEQ ID NO:1, and/or amino acids 243-251 of SEQ ID NO:3.

In another embodiment, the invention relates to a FLINT analogcomprising an amino acid substitution (s) in the region comprising aminoacids 214-222 of SEQ ID NO:1, selected from the group consisting of:

a. Pro at position 215 is replaced by any naturally occurring amino acidother than Pro;

b. Thr at position 216 is replaced by any naturally occurring amino acidother than Thr;

c. Pro at position 217 is replaced by any naturally occurring amino acidother than Pro;

d. Arg at position 218 is replaced by any naturally occurring amino acidother than Arg;

e. Ala at position 219 is replaced by any naturally occurring amino acidother than Ala;

f. Gly at position 220 is replaced by any naturally occurring amino acidother than Gly;

g. Arg at position 221 is replaced by any naturally occurring amino acidother than Arg;

h. Ala at position 222 is replaced by any naturally occurring amino acidother than Ala.

In another embodiment, the invention relates to a FLINT analogcomprising an amino acid substitution in the region comprising aminoacids 214-222 of SEQ ID NO:1, selected from the group consisting of:

a. Gly at position 214 is replaced by a positively charged amino acidthat is not Gly;

b. Pro at position 215 is replaced by a positively charged amino acidthat is not Pro;

c. Thr at position 216 is replaced by a positively charged amino acidthat is not Thr;

d. Pro at position 217 is replaced by a positively charged amino acidthat is not Pro;

e. Arg at position 218 is replaced by a positively charged amino acidthat is not Arg;

f. Ala at position 219 is replaced by a positively charged amino acidthat is not Ala;

g. Gly at position 220 is replaced by a positively charged amino acidthat is not Gly;

h. Arg at position 221 is replaced by a positively charged amino acidthat is not Arg;

i. Ala at position 222 is replaced by a positively charged amino acidthat is not Ala.

In another embodiment, the invention relates to a FLINT analogcomprising an amino acid substitution in the region comprising aminoacids 214-222 of SEQ ID NO:1, selected from the group consisting of:

a. Gly at position 214 is replaced by a negatively charged amino acidthat is not Gly;

b. Pro at position 215 is replaced by a negatively charged amino acidthat is not Pro;

c. Thr at position 216 is replaced by a negatively charged amino acidthat is not Thr;

d. Pro at position 217 is replaced by a negatively charged amino acidthat is not Pro;

e. Arg at position 218 is replaced by a negatively charged amino acidthat is not Arg;

f. Ala at position 219 is replaced by a negatively charged amino acidthat is not Ala;

g. Gly at position 220 is replaced by a negatively charged amino acidthat is not Gly;

h. Arg at position 221 is replaced by a negatively charged amino acidthat is not Arg;

i. Ala at position 222 is replaced by a negatively charged amino acidthat is not Ala.

In another embodiment, the invention relates to a FLINT analogcomprising an amino acid substitution in the region comprising aminoacids 214-222 of SEQ ID NO:1, selected from the group consisting of:

a. Gly at position 214 is replaced by a polar uncharged amino acid thatis not Gly;

b. Pro at position 215 is replaced by a polar uncharged amino acid thatis not Pro;

c. Thr at position 216 is replaced by a polar uncharged amino acid thatis not Thr;

d. Pro at position 217 is replaced by a polar uncharged amino acid thatis not Pro;

e. Arg at position 218 is replaced by a polar uncharged amino acid thatis not Arg;

f. Ala at position 219 is replaced by a polar uncharged amino acid thatis not Ala;

g. Gly at position 220 is replaced by a polar uncharged amino acid thatis not Gly;

h. Arg at position 221 is replaced by a polar uncharged amino acid thatis not Arg;

i. Ala at position 222 is replaced by a polar uncharged amino acid thatis not Ala.

In another embodiment, the invention relates to a FLINT analogcomprising an amino acid substitution in the region comprising aminoacids 214-222 of SEQ ID NO:1, selected from the group consisting of:

a. Gly at position 214 is replaced by a nonpolar amino acid that is notGly;

b. Pro at position 215 is replaced by a nonpolar amino acid that is notPro;

c. Thr at position 216 is replaced by a nonpolar amino acid that is notThr;

d. Pro at position 217 is replaced by a nonpolar amino acid that is notPro;

e. Arg at position 218 is replaced by a nonpolar amino acid that is notArg;

f. Ala at position 219 is replaced by a nonpolar amino acid that is notAla;

g. Gly at position 220 is replaced by a nonpolar amino acid that is notGly;

h. Arg at position 221 is replaced by a nonpolar amino acid that is notArg;

i. Ala at position 222 is replaced by a nonpolar amino acid that is notAla.

In another embodiment, the invention relates to a FLINT analogcomprising an amino acid substitution in the region comprising aminoacids 214-222 of SEQ ID NO:1, selected from the group consisting of:

a. Arg at position 218 is replaced by Gln;

b. Arg at position 218 is replaced by Glu;

c. Thr at position 216 is replaced by Pro;

d. Arg at position 218 is replaced by Ala;

e. Arg at position 218 is replaced by Gly;

f. Arg at position 218 is replaced by Ser;

g. Arg at position 218 is replaced by Val

h. Arg at position 218 is replaced by Tyr;

i. Pro at position 217 is replaced by Tyr

j. Thr at position 216 is replaced by Pro, and Arg at position 218 isreplaced by Gln.

In another embodiment, the present invention relates to a FLINT analogcomprising SEQ ID NO:1 wherein Arg at position 34 is replaced by Asn,Asp at position 36 is replaced by Thr, and Arg at position 218 isreplaced by Gln, Glu, Ala, Gly, Ser. Val, or Tyr.

In another embodiment, the present invention relates to a FLINT analogcomprising SEQ ID NO:1 wherein Arg at position 34 is replaced by Asn,Asp at position 36 is replaced by Thr, Asp at position 194 is replacedby Asn, Ser at position 196 is replaced by Thr, and Arg at position 218is replaced by Gln, Glu, Ala, Gly, Ser, Val, or Tyr.

In another embodiment, the present invention relates to a FLINT analogcomprising one or more amino acid substitution(s) within SEQ ID NO:1wherein Arg at position 34 is replaced by Asn, Asp at position 36 isreplaced by Thr, and Arg at position 218 is replaced by an amino acidselected from the group consisting of:

a. any naturally occurring amino acid that is not Arg;

b. any positively charged amino acid that is not Arg;

c. any negatively charged amino acid that is not Arg;

d. any polar uncharged amino acid that is not Arg;

e. any nonpolar amino acid that is not Arg; and

f. an amino acid that is Glu, Gln, Ala, Gly, Ser, Val, or Tyr.

In another embodiment, the present invention relates to a FLINT analogcomprising one or more amino acid substitutions within SEQ ID NO:1wherein Arg at position 34 is replaced by Asn, Asp at position 36 isreplaced by Thr, Asp at position 194 is replaced by Asn, Ser at position196 is replaced by Thr, and Arg at position 218 is replaced by an aminoacid selected from the group consisting of:

a. any naturally occurring amino acid that is not Arg;

b. any positively charged amino acid that is not Arg;

c. any negatively charged amino acid that is not Arg;

d. any polar uncharged amino acid that is not Arg;

e. any nonpolar amino acid that is not Arg; and

f. an amino acid that is Glu, Gln, Ala, Gly, Ser, Val, or Tyr.

In another embodiment, the present invention relates to a FLINT analogcomprising one or more amino acid substitution(s) within SEQ ID NO:1wherein Ser at position 132 is replaced by Asn, and Arg at position 218is replaced by an amino acid selected from the group consisting of:

a. any naturally occurring amino acid that is not Arg;

b. any positively charged amino acid that is not Arg;

c. any negatively charged amino acid that is not Arg;

d. any polar uncharged amino acid that is not Arg;

e. any nonpolar amino acid that is not Arg; and

f. an amino acid that is Glu, Gln, Ala, Gly, Ser, Val, or Tyr.

Another embodiment relates to a nucleic acid encoding aprotease-resistant FLINT analog of the present invention.

In another embodiment, the invention relates to a protease resistantFLINT analog that is encoded by a nucleic acid that hybridizes to SEQ IDNO:2 under high stringency conditions.

In another embodiment, the present invention relates to a nucleic acidthat encodes a protease resistant FLINT analog, said nucleic acidhybridizing to SEQ ID NO:2 under high stringency conditions.

In another embodiment, the present invention relates to a vectorcomprising a nucleic acid encoding a protease-resistant FLINT analog.

In another embodiment the invention relates to therapeutic and clinicaluses of a protease resistant FLINT analog to prevent or treat a diseaseor condition in a mammal in need of such prevention or treatment.

In another embodiment the invention relates to therapeutic and clinicaluses of a protease resistant FLINT analog to prevent or treat acute lunginjury (ALI), acute respiratory distress syndrome (ARDS), ulcerativecolitis, and to facilitate organ preservation for transplantation.

In another embodiment the present invention relates to a pharmaceuticalcomposition comprising a protease resistant FLINT analog.

In another embodiment the present invention relates to the use of FLINTanalog to inhibit T lymphocyte activation.

In another embodiment, the present invention relates to the use of FLINTanalog to prevent or treat chronic obstructive pulmonary disease (COPD).

In another embodiment, the present invention relates to the use of FLINTanalog to prevent or treat pulmonary fibrosis (PF).

In another embodiment, the present invention relates to a method forproducing a protease resistant FLINT analog, said analog being resistantto proteolysis by a trypsin-like protease between positions 218 and 219of SEQ ID NO:1 (alternatively between positions 247 and 248 of SEQ IDNO:3), comprising the step of altering the amino acid sequence in theregion at and/or between positions 214 to 222 of SEQ ID NO:1, asdescribed herein.

DETAILED DESCRIPTION OF THE INVENTION

SEQ ID NO:1—Mature human FLINT, i.e. native FLINT minus the leadersequence.

SEQ ID NO:2—Nucleic acid/cDNA encoding mature human FLINT.

SEQ ID NO:3—Native human FLINT.

SEQ ID NO:4—Human FLINT leader sequence.

SEQ ID NO:5—Oligonucleotide primer A, CF107

SEQ ID NO:6—Oligonucleotide primer B, CF111

SEQ ID NO:7—Oligonucleotide primer C, CF112

SEQ ID NO:8—Oligonucleotide primer D, CF110

SEQ ID NO:9—Nucleic acid/cDNA encoding native human FLINT.

FIG. 1. Time course thrombin cleavage of native FLINT and R218Q analog.

FIG. 2. FLINT analog R218Q inhibits FasL-induced apoptosis in Jurkatcells. FLINT samples purified from AV12 cells.

FIG. 3. FLINT analog R218Q purified from 293 EBNA cells inhibits FasLinduced apoptosis in Jurkat cells.

FIG. 4. FLINT analog RDDSR inhibits FasL-induced apoptosis in Jurkatcells.

FIG. 5. RP-HPLC profile of radioactivity after an in vitro incubation of¹²⁵I-FLINT and ¹²⁵I-FLINT(R218Q) with ICR mouse blood. Test articleswere incubated for 1 h at 37° C. Serum was prepared and fractionated byRP-HPLC. Data is expressed as the percentage of radioactivity perfraction applied to the column.

FIG. 6. RP-HPLC profile of FLINT immunoreactivity in plasma 15 min.after an intravenous administration of FLINT, or FLINT (R218Q), to ICRmice. Fractions were collected and analyzed by ELISA. Data arerepresentative of findings from each individual animal.

FIG. 7. FLINT and FLINT analog dose response in mouse acute liverfailure (ALF) model.

The term “analog” or “FLINT analog” means a variant FLINT, or fragmentthereof, resistant to proteolysis between positions 218 and 219 of SEQID NO:1 (positions 247 and 248 of SEQ ID NO:3), and which preferably hasbiological activity substantially the same as FLINT.

The term “negatively charged group” or “negatively charged amino acid”refers to Asp or Glu.

The term “positively charge group” or “positively charged amino acid”refers to His, Arg, or Lys.

The term “polar uncharged” or “polar uncharged amino acid” refers toCys, Thr, Ser, Gly, Asn, Gln, and Tyr.

The term “nonpolar” or “nonpolar amino acid” refers to Ala, Pro, Met,Leu, Ile, Val, Phe, or Trp.

The term “naturally-occurring amino acid” refers to any of the 20L-amino acids that are found in proteins.

The term “native FLINT” refers to SEQ ID NO:3.

The term “mature FLINT” refers to SEQ ID NO:1.

The term “FLINT” refers to native and mature FLINT from human, otherprimates, and other mammalian and non-mammalian sources.

As used herein “half-life” refers to the time required for approximatelyhalf of FLINT or FLINT analog molecules in a sample to beproteolytically cleaved between positions 218 and 219 of SEQ ID NO:1, invitro and/or in vivo, as determined by any suitable means.

The term oprotease-resistantm or “resistant” refers to a FLINT analogthat, when compared with FLINT, or FLINT fragment, is more resistant toproteolysis between residues 218 and 219 of SEQ ID NO:1. Proteaseresistant analogs differ from FLINT by one or more amino acidsubstitutions, deletions, inversions, additions, and/or changes inglycosylation sites, or patterns, as compared with or against nativeFLINT, or mature FLINT, or other FLINT fragment. Preferably thesechanges occur in the region from about position 214 through position 222of SEQ ID NO:1.

The term “protease-resistant” contemplates degrees of resistance toproteolysis at position 218 from complete resistance to partialresistance. Thus, a “substantially resistant” analog shows a degree ofresistance to proteolysis at position 218, for example, an analog with ahalf-life that is at least about 25% greater than native FLINT whentreated or exposed to a suitable protease. Preferably, a substantiallyresistant FLINT analog possesses a protease resistance half-life that isat least about 2-fold greater than native FLINT.

Susceptibility to proteolysis will depend on factors such as the aminoacid sequence at or near the site of cleavage and/or the recognitionsite for the particular proteolytic enzyme involved, and on the physicaland chemical environment of the particular analog under consideration.Such factors can affect the K_(M) and/or rate of proteolysis by aproteolytic enzyme.

The recognition site for serine proteases including thrombin has beeninvestigated. Thrombin will cleave at multiple sites including LVPR/ andsites related to LVPR/, e.g. VDPR/ as well as others. Charge density andsteric properties operative at an enzyme's active site will determinethe degree to which proteolysis occurs. Thus, the present inventioncontemplates protease resistant analogs of FLINT that comprise one ormore amino acid substitutions, deletions, or additions within a windowdefined by residues 215 through 218 of SEQ ID NO:1. Such analogs areeasily constructed by the skilled artisan using known recombinanttechniques and testable in vitro for resistance to proteolysis atposition 218. All such embodiments are intended to be within the scopeof the present invention.

Protease resistance, as contemplated herein, refers to the sensitivityof a FLINT analog to proteolysis at position 218, in vivo or in vitro.For example, the resistance of an analog to a trypsin-like protease suchas thrombin or trypsin, or other serine protease is compared with theresistance shown by FLINT under the same conditions. It is preferredthat a FLINT analog display a half-life at least 5% greater than FLINT,alternatively at least 10%, 20%, 30%, 40%, or between 50% to 100%greater than wild type FLINT, as determined by the relative quantity offull length molecules to smaller digestion products (e.g. fragments1-218 and 219-271 of SEQ ID NO:1). Any suitable method for making aqualitative and/or quantitative assessment of said relative quantitiescan be used, for example, polyacrylamide gel electrophoresis. Mostpreferably, a resistant analog possesses a half-life that is from about1-fold to 2-fold greater than FLINT to about 100-fold or greater thanFLINT.

“Fragment thereof” refers to a fragment, piece, or sub-region of a FLINTnucleic acid or protein molecule or analog of the present invention,such that the fragment comprises 5 or more amino acids, or 10 or morenucleotides that are contiguous in the parent protein or nucleic acidmolecule.

The term “fusion protein” denotes a hybrid protein molecule not found innature comprising a translational fusion or enzymatic fusion in whichtwo or more different proteins or fragments thereof are covalentlylinked on a single polypeptide chain.

“Functional fragment” or functionally equivalent fragment, as usedherein, refers to a region, or fragment of a full length protein of thepresent invention that are capable of providing a substantially similarbiological activity as a full length protein of the invention, in vivoand/or in vitro, viz. the capacity to inhibit apoptosis. Functionalfragments may be produced by cloning technology, or as the naturalproducts of alternative splicing mechanisms.

“Host cell” refers to any eucaryotic or procaryotic cell that issuitable for propagating and/or expressing a cloned gene or nucleic acidcontained on a vector that is introduced into said host cell by, forexample, transformation or transfection, or the like.

The term “hybridization” as used herein refers to a process in which asingle-stranded nucleic acid molecule joins with a complementary strandthrough nucleotide base pairing. “Selective hybridization” refers tohybridization under conditions of high stringency. The degree ofhybridization depends upon the degree of homology, the stringency ofhybridization, and the length of hybridizing strands.

“Isolated nucleic acid compound” refers to any RNA or DNA sequence,however constructed or synthesized, which is locationally distinct fromits natural location.

The term “stringency” refers to hybridization conditions. Highstringency conditions disfavor non-homologous base pairing. Lowstringency conditions have the opposite effect. Stringency may bealtered, for example, by temperature and salt concentration.

“Low stringency” conditions comprise, for example, a temperature ofabout 37° C. or less, a formamide concentration of less than about 50%,and a moderate to low salt (SSC) concentration; or, alternatively, atemperature of about 50° C. or less, and a moderate to high salt (SSPE)concentration, for example 1M NaCl.

“High stringency” conditions are well known to the skilled artisan andcan comprise, for example, a temperature of about 42° C. or less, aformamide concentration of less than about 20%, and a low salt (SSC)concentration; or, alternatively, a temperature of about 65° C., orless, and a low salt (SSPE) concentration. For example, high stringencyconditions comprise hybridization in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C. (See e.g. Ausubel, F. M. et al.Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. NewYork, at 2.10.3).

“SSC” comprises a hybridization and wash solution. A stock 20×SSCsolution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.

“SSPE” comprises a hybridization and wash solution. A 1×SSPE solutioncontains 180 mM NaCl, 9 mM Na₂HPO₄, 0.9 mM NaH₂PO₄ and 1 mM EDTA, pH7.4.

“Substantially pure” used in reference to a peptide or protein meansthat said peptide or protein is separated from a large fraction of allother cellular and non-cellular molecules, including other proteinmolecules. A substantially pure preparation would be about at least 85%pure; preferably about at least 95% pure. For example, a “substantiallypure” protein as described herein could be prepared by the IMAC proteinpurification method.

The term “vector” as used herein refers to a nucleic acid compound usedfor introducing exogenous or endogenous DNA into host cells. A vectorcomprises a nucleotide sequence, which may encode one or more proteinmolecules. Plasmids, cosmids, viruses, and bacteriophages, in thenatural state or which have undergone recombinant engineering, areexamples of commonly used vectors.

The nucleotide and amino acid abbreviations used herein are thoseaccepted in the art and by the United States Patent and TrademarkOffice, as set forth in 37 C.F.R. 1.822 (b)(2).

Descriptions herein relating to proteolysis of FLINT and FLINT analogsbetween positions 218 and 219 of SEQ ID NO:1 (mature FLINT) are intendedalso to relate to SEQ ID NO:3 (native FLINT with leader), where thecomparable region lies between positions 247 and 248.

Applicants have discovered that FLINT polypeptides are cleaved in vivobetween the arginine residue at position 218 and the alanine residue atposition 219 of SEQ ID NO:1, probably by a trypsin-like protease. Acleavage product of this reaction comprises residues 1-218 of SEQ IDNO:1, termed “FLINT metabolite.” FLINT metabolite can be produced invitro by treating a FLINT polypeptide with a trypsin-like protease, forexample, thrombin, trypsin, or other serine protease.

One embodiment of the present invention relates to a method to produceanalogs of a FLINT polypeptide that are resistant to proteolysis betweenpositions 218 and 219 of SEQ ID NO:1 and retain biological activity.Biological activity relates to the capacity of an analog to bind FasLand/or LIGHT, and may include an inhibition of apoptosis in viva and/orin vitro.

Another embodiment of the present invention relates to analogs of aFLINT polypeptide that are resistant to proteolysis between positions218 and 219 of SEQ ID NO:1 and retain biological activity. Biologicalactivity relates to the capacity of an analog to bind FasL and/or LIGHT,and may include an inhibition of apoptosis in vivo and/or in vitro.

Preferred FLINT analogs provide a half-life at least 5%, 10%, 20%, 30%,40%, or between 50% to 100% greater than FLINT, as determined by theratio over time of full length FLINT to digestion products comprisingFLINT metabolite and the carboxyl fragment (i.e. residues 219-271 of SEQID NO1); most preferably a FLINT analog possesses a half-life at least2-fold to 100-fold or greater than FLINT.

FLINT analogs comprise one or more primary or secondary structuralchanges, for example amino acid substitutions, deletions, inversions,additions, or changes in glycosylation sites or patterns and/orcombinations thereof that prevent or diminish proteolysis, and/or therate thereof, between positions 218 and 219 of SEQ ID NO:1. Preferablythese changes occur at or near the thrombin-like recognition sequence,in the case of FLINT, PTPR; most preferably, at or near the PR dipeptidesequence at positions 217 and 218 of SEQ ID NO:1. As the skilled artisanunderstands, residues at or near a recognition site can also affect thesusceptibility of the substrate protein to proteolysis by altering thecharge milieu at the active site and/or by creating alterations bysteric hindrance in the region of the active site.

Therefore, the invention contemplates FLINT analogs comprising aminoacid changes in FLINT, preferably in the region from about position 214through position 222 of SEQ ID NO:1 or the comparable region of SEQ IDNO:3, wherein said analogs are resistant to proteolysis at position 218of SEQ ID NO:1.

Also contemplated are protease-resistant FLINT analogs comprisingsubstitutions, deletions, insertions, inversions, additions, or changesin glycosylation sites or patterns that occur outside the preferredwindow comprising residues 214 through 222 of SEQ ID NO:1. As theskilled artisan understands, many substitutions, and/or other changes ina protein's sequence or structure, can be made without substantiallyaffecting the biological activity of the protein. For example, makingconservative amino acid substitutions, or changing one amino acid foranother from the same class of amino acids, for example negativelycharged residues, positively charged residues, polar uncharged residues,and non-polar residues, or any other classification acceptable in theart are often without effect on function. Such changes are intended tobe within the scope of the present invention.

In one embodiment, a single amino acid change is made within thisregion; alternatively, at least two changes are made within this region;alternatively, at least three changes are made within this region;alternatively, at least four changes are made within this region.

In one embodiment, the invention relates to FLINT analog polypeptidesand nucleic acids that are defined with reference to a percent identitysimilarity to SEQ ID NO:1, SEQ ID NO:2, and/or SEQ ID NO:3. Sequenceidentity refers to a comparison between two molecules using standardalgorithms well known in the art. Although any suitable sequencecomparison algorithm can be used for this purpose, for illustration,this embodiment shall be described with reference to the well-knownSmith-Waterman algorithm using SEQ ID NO:1 as the reference sequence todefine percent identity to a comparator sequence. When sequence identityis used with reference to a polypeptide, the entire polypeptide may beused in the comparison or a defined sub-region thereof.

The choice of parameter values for matches, mismatches, and inserts ordeletions is arbitrary. A preferred set of values for use with theSmith-Waterman algorithm is set forth in the “maximum similaritysegments” approach, which uses values of 1 for a matched residue, and−1/3for a mismatched residue (See Waterman, Bulletin of MathematicalBiology, 46, 473-500, 1984). Insertions and deletions (indels), x, areweighted as follows:

X _(k)=1+k/3

Where k is the number of residues in a given insert or deletion.

For example, a comparator sequence that has 20 substitutions and 3insertions relative to the 250 residue reference protein sequence wouldhave an identity of:

[(1×250)−(1/3×20)−(1+3/3)]/250=96%

identical.

FLINT analogs of the present invention can easily be tested forbiological activity and/or sensitivity to proteolysis as describedherein; See e.g. Examples 11 and 12. Biological activity can be assessedusing either in vitro (see Example 6) or in vivo (see Example 9) modelsas described herein.

FLINT analogs are active in binding FasL and/or LIGHT. LIGHT, a memberof the TNF family, is a membrane-bound ligand that triggers distinctbiological responses. LIGHT may play a role in immune modulation, and itappears to be involved in herpes virus entry (see Zhai et al., J. Clin.Invest. 102, 1142-1151, 1998; Montgomery et al. Cell, 87, 427-436,1996). Soluble LIGHT inhibits the proliferation of various tumor cellsand appears to bind the receptors LTPR and TR2 (also referred to asherpes virus entry mediator, IWEM). LIGHT is expressed highly inactivated lymphocytes and evokes immune modulation from hematopoieticcells. For example, LIGHT stimulates the secretion of IFNγ. LIGHT alsoinduces apoptosis of tumor cells that express the LTβR and TR2/HVEMreceptors. The cytotoxic effect of LIGHT, which is enhanced by IFNγ, canbe blocked by addition of soluble LTβR-Fc or TR2/HVEM-Fc.

The present invention relates further to the use of FLINT analog to bindLIGHT, thereby inhibiting T cell activation. T cell activation can bechronically suppressed when advantageous, for example, following organtransplantation to prevent rejection, in the treatment of autoimmunediseases, and in treating systemic inflammatory responses.

LIGHT is produced primarily by activated T lymphocytes. When LIGHT bindsto HVEM on the surface of T cells it stimulates T cell proliferation (J.A. Harrop et al. J. Biol. Chem. 273, 27548-27556, 1998).

FLINT analogs of the invention can be produced by recombinant techniquesor by direct chemical synthesis. The analogs may also be produced byrecombinant DNA mutagenesis techniques, well known to the skilledartisan. See. e.g. K. Struhl, “Reverse biochemistry: Methods andapplications for synthesizing yeast proteins in vitro,” Meth. Enzymol.194, 520-535. In a preferred recombinant method, site-directedmutagenesis is used to introduce defined changes into the region 214-222of SEQ ID NO:1 or the comparable region of SEQ ID NO:3.

FLINT analogs also include modified derivatives thereof in which one ormore polyethylene glycol groups (hereinafter “PEG” groups) are bonded tothe N-terminus or to amine groups or thiol groups in the amino acid sidechain(s). Suitable PEG groups generally have a molecular weight betweenabout 5000 and 20,000 atomic mass units. Procedures for preparingPEGylated polypeptides are disclosed in Mumtaz and Bachhawat, IndianJournal of Biochemistry and Biophysics 28:346 (1991) and Franciset al.,International Journal of Hematology 68:1 (1998), the entire teachings ofwhich are incorporated herein by reference.

Yet another embodiment of a FLINT analog is a molecule comprising two ormore modified or unmodified FLINT analogs, e.g., a dimerized FLINTanalog such as R218Q. Homodimers comprising two identical analogsubunits (e.g. R218Q(2)), and heterodimers, comprising two non-identicalanalog subunits (e.g. R218Q/R34N, D36T, D194N, S196T, R218Q) arecontemplated. Dimerization can be accomplished using the PEG polymerchain method as described in Espat et al., Journal of Surgical Research59: 153 (1995), or through a C-terminal fusion to a domain that inducesdimerization such as a leucine zipper, as described in O'Shea et al.,Science 254:539 (1991). The entire teachings of Espat and O'Shea areincorporated herein by reference.

Another embodiment of the invention relates to a fusion proteincomprising a FLINT analog. “Fusion protein” denotes a hybrid proteinmolecule not found in nature comprising a translational fusion orenzymatic fusion in which two or more different proteins or fragmentsthereof are covalently linked on a single polypeptide chain. Human serumalbumin and the C-terminal domain of thrombopoietin are examples ofproteins which could be fused with a FLINT analog. Procedures forpreparing fusion proteins are disclosed in EP394,827, Tranecker et al.,Nature 331:84 (1988) and Fares, et al., Proc. Natl. Acad. Sci. USA89:4304 (1192), the entire teachings of which are incorporated herein byreference.

A FLINT analog fusion protein comprises two protein segments fusedtogether by means of a peptide bond. The first protein segment consistsof at least 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 50, 75, 100,125, 150, 175, 200, 225, 250, or 275 contiguous amino acid residues ofan analog of the present invention (i.e. protease resistant SEQ ID NO:1or SEQ ID NO:3). The first protein can alternatively be a full lengthanalog of the present invention and can be N-terminal or C-terminal.

The second protein of a FLINT analog fusion protein can be a full lengthprotein or a protein fragment. Proteins commonly used in fusion proteinsinclude B-galactosidase, B-glucuronidase, green fluorescent protein(GFP), thrombopoietin (TPO), glutathione-S-transferase (GST),luciferase, horseradish peroxidase, and chloramphenicolacetyltransferase (CAT). Epitope tags can be used in fusion proteinconstructions including histidine (His) tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin tags.Other fusion constructions can include maltose binding protein, S-tag,Lex A DNA binding domain, GAL4 DNA binding domain fusions, and herpessimplex virus BP16 protein fusions.

The FLINT analogs of the present invention can be glycosylated orunglycosylated. A glycosylated polypeptide is modified with one or moremonosaccharides or oligosaccharides. A monosaccharide is a chiralpolyhydroxyalkanol or polyhydroxyalkanone which typically exists inhemiacetal form. An “oligosaccharide” is a polymer of from about 2 toabout 10 monosaccharides which are generally linked by acetal bonds. Onetype of glycosyl group commonly found in glycosylated proteins isN-acetylneuraminic acid. A glycosylated polypeptide can beN-glycosylated and/or O-glycosylated, preferably N-glycosylated.

The term “N-glycosyled polypeptide” refers to polypeptides having one ormore NXS/T motifs in which the nitrogen atom in the side chain amide ofthe asparagine is covalently bonded to a glycosyl group. “X” refers toany naturally occurring amino acid residue except proline. The“naturally occurring amino acids” are glycine, alanine, valine, leucine,isoleucine, proline, serine, threonine, cysteine, methionine, lysine,arganine, glutamic acid, asparatic acid, glutamine, asparagine,phenylalanine, histidine, tyrosine and tryptophan. N-Glycosylatedproteins are optionally O-glycosylation.

The term “O-glycosyled polypeptide” refers to polypeptides having one ormore serines and/or threonine in which the oxygen atom in the side chainis covalently bonded to a glycosyl group. O-Glycosylated proteins areoptionally N-glycosylation.

Glycosylated polypeptides and analogs of the invention can be preparedrecombinantly by expressing a gene encoding a polypeptide in a suitablemammalian host cell, resulting in glycosylation of side chain amidesfound in accessible NXT/S motifs on the polypeptide surface and of sidechain alcohols of surface accessible serines and threonines. Specificprocedures for recombinantly expressing genes in mammalian cells areprovided hereinbelow. Other procedures for preparing glycosylatedproteins are disclosed in EP 640,619 to Elliot and Burn, the entireteachings of which are incorporated herein by reference. Unglycosylatedpolypeptides can be prepared recombinantly by expressing a gene encodinga polypeptide in a suitable procaryotic host cell.

The present invention also relates to nucleic acids, e.g. cDNAs, DNAS,or RNAs, encoding a FLINT analog of the present invention and vectorscomprising said nucleic acids. The skilled artisan understands that saidnucleic acids can be prepared synthetically by mutating a nucleic acidtemplate that encodes FLINT, e.g. introducing appropriate pointmutations into a CDNA encoding FLINT using any number of suitablemutagenic techniques known to the skilled artisan to produce a proteaseresistant or substantially protease resistant analog of the presentinvention. Alternatively, said nucleic acids can be preparedsynthetically de novo based on knowledge of the genetic code and theparticular analog of SEQ ID NO:1 or SEQ ID NO:3 that one is interestedin. Codon preference may be taken into account when designing a suitablenucleic acid.

For example, the amino acid sequences of the analogs of the presentinvention are described elsewhere in this application as comprisingdeletions, substitutions and/or additions at and around position 218 ofSEQ ID NO:1, or elsewhere in the FLINT sequence. The skilled artisanunderstands that use of the genetic code in combination with knowledgeof preferred codon usage is adequate to describe and would enable theconstruction of any number of nucleic acids that encode any particularanalog desired. For example, the codon that encodes arginine at position218 of SEQ ID NO:1 in native FLINT is “agg.” One of the analogs of theinvention changes the arginine at this position to glutamine, which maybe accomplished by changing the codon from “agg” to “caa” or “cag.”Choice of which codon to use for any particular amino acid may be basedon knowledge of preferred codon usage and/or by trial and errorconsidering expression of the analog. Other analogs contemplated by theinvention are describable and enabled in the same manner.

A FLINT CDNA can be synthesized by RT-PCR using conventional techniques.For example, PolyA RNA is prepared from a tissue known to express theFLINT gene (e.g. human lung), using standard methods. First strand FLINTCDNA synthesis is achieved in a reverse transcriptase reaction using aFLINT sequence derived downstream primer. A commercially available kitsuch as GEN MP by Perkin Elmer may be employed. In a subsequent PCR,FLINT specific forward and reverse primers are used to amplify the CDNA.The amplified sample may be analyzed by agarose gel electrophoresis tocheck the length of the amplified fragment.

FLINT CDNA generated in this manner is used as a template forintroducing appropriate point mutations (i.e. construction of FLINTanalog cDNAs). A suitable protocol is described in detail in “CurrentProtocols in Molecular Biology”, volume 1, section 8.5.7 (John Wiley andSons, Inc. publishers), the entire teachings of which are incorporatedherein by reference. Briefly, synthetic oligonucleotides are designed toincorporate one or more point mutations) at one end of an amplifiedfragment, e.g. at position 218 of SEQ ID NO:1. Following first strandPCR, the amplified fragments encompassing the mutation are annealed witheach other and extended by mutually primed synthesis. Annealing isfollowed by a second PCR step utilizing 5′ forward and 3′ reverse endprimers in which the entire mutagenized fragment gets amplified and isready for subdloning into the appropriate vector.

The skilled artisan understands that the degeneracy of the genetic codeprovides multiple codons in some instances for a given amino acid. Allsuch nucleic acid sequence variants are intended to be within the scopeof the invention.

Using the information provided herein, such as the amino acid sequenceof SEQ ID NO:1 or SEQ ID NO:3, or the nucleotide sequence of SEQ ID NO:2or variants thereof, a nucleic acid molecule of the present inventionencoding a FLINT analog can be obtained using well-known methods.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, or any other form, or in the form of DNA,including, but not limited to, CDNA and genomic DNA obtained by cloningor produced synthetically, or any combination thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

The present invention further provides isolated nucleic acids thatencode a protease resistant FLINT analog and that hybridize underselective conditions to a polynucleotide disclosed and/or contemplatedherein, e.g., SEQ ID NO:2 and/or derivatives thereof.

The present invention further provides isolated nucleic acids comprisingFLINT analog polynucleotides, wherein the polynucleotides arecomplementary to the polynucleotides of the invention. Complementarysequences base-pair throughout the entirety of their length with suchpolynucleotides (i.e., have 100% sequence identity over their entirelength). Complementary bases associate through hydrogen bonding indouble-stranded nucleic acids. For example, the following base pairs arecomplementary: guanine and cytosine; adenine and thymine; and adenineand uracil. (See, e.g., Ausubel, supra; or Sambrook, supra)

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, (c)purification techniques, or combinations thereof, as well known in theart.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. For example, a hexa-histidine marker sequence provides aconvenient means to purify the proteins of the present invention.

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or a hybrid thereof, can be obtained from biologicalsources using any number of cloning methodologies known to those ofskill in the art. In some embodiments, oligonucleotide probes whichselectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a CDNA or genomic DNA library. Isolation of RNA andconstruction of cDNA and genomic libraries is well known to those ofordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes may be used to hybridize with genomic DNA orCDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled bytemperature, ionic strength, pH and the presence of a partiallydenaturing solvent such as formamide. For example, the stringency ofhybridization is conveniently varied by changing the polarity of thereactant solution through, for example, manipulation of theconcentration of formamide within the range of 0% to 50%. The degree ofcomplementarity (sequence identity) required for detectable binding willvary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%; however, it should be understood that minor sequence variations inthe probes and primers may be compensated for by reducing the stringencyof the hybridization and/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification.processes(see, e.g., U.S. Pat. Nos. U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159, 4,965,188, to Mullis, et al.; U.S. Pat. Nos. 4,795,699 and4,921,794 to Tabor, et al; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat.No. 5,122,464 to Wilson, et al.; U.S. Pat. No. 5,091,310 to Innis; U.S.Pat. No. 5,066,584 to Gyllensten, et al; U.S. Pat. No. 4,889,818 toGelfand, et al; U.S. Pat. No. 4,994,370 to Silver, et al; U.S. Pat. No.4,766,067 to Biswas; U.S. Pat. No. 4,656,134 to Ringold) and RNAmediated amplification which uses anti-sense RNA to the target sequenceas a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238to Malek, et al, with the tradename NASBA), the entire contents of whichare herein incorporated by reference. (See, e.g., Ausubel, supra; orSambrook, supra)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA, cDNA libraries, or cloned DNAor RNA. PCR and other in vitro amplification methods may also be useful,for example, to clone nucleic acid sequences that code for proteins tobe expressed, to make nucleic acids to use as probes for detecting thepresence of the desired mRNA in samples, for nucleic acid sequencing, orfor other purposes. Examples of techniques sufficient to direct personsof skill through in vitro amplification methods are found in Berger,Sambrook, and Ausubel, as well as Mullis, et al., U.S. Pat. No.4,683,202 (1987); and Innis, et al., PCR Protocols A Guide to Methodsand Applications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech). The T4 gene32 protein (Boehringer Mannheim) can be used to improve yield of longPCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., Meth. Enzymol. 68:90-99 (1979); thephosphodiester method of Brown, et al., Meth. Enzymol. 68:109-151(1979); the diethylphosphoramidite method of Beaucage, et al., Tetra.Letts. 22:1859-1862 (1981); the solid phase phosphoramidite triestermethod described by Beaucage and Caruthers, Tetra. Letts.22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter, et al., Nucleic Acids Res.12:6159-6168 (1984); and the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis generally produces a single-strandedoligonucleotide, which may be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences may beobtained by the ligation of shorter sequences.

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and production ofFLINT analogs, as is well known in the art. (See, e.g., Sambrook, etal., 1989; Ausubel, et al., 1987-1998, which is entirely incorporatedherein by reference).

“Vector” refers to a nucleic acid compound used for introducingexogenous or endogenous nucleic acid into host cells. A vector comprisesa polydeoxynucleotide sequence which encodes a FLINT analog or fusionprotein thereof. Plasmids, cosmids, viruses and bacteriophages, in anatural state or which have undergone recombinant engineering, arenon-limiting examples of commonly used vectors to provide recombinantvectors comprising at least one desired isolated nucleic acid molecule.

“Host cell” refers to any eucaryotic, procaryotic, or other cell orpseudo cell or membrane-containing construct that is suitable forpropagating and/or expressing an isolated nucleic acid that isintroduced into a host cell by any suitable means known in the art(e.g., transformation or transfection, or the like), or induced toexpress an endogenous polydeoxynucleic acid. The cell can be part of atissue or organism, isolated in culture or in any other suitable form.

The nucleic acids of the invention, including cDNAs, can optionally bejoined to a vector containing a selectable marker for propagation in ahost. Generally, a plasmid vector is introduced in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a charged lipid(e.g., lipofectamine). If the vector is a virus, it can be packaged invitro using an appropriate packaging cell line and then transduced intohost cells.

The DNA insert may be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, phage T7 promoter, the E. colilac, tzp and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, or any other suitable promoter. Othersuitable promoters will be known to the skilled artisan. The expressionconstructs will further contain sites for transcription initiation,termination and, in the transcribed region, a ribosome binding site fortranslation. The coding portion of the mature transcripts expressed bythe constructs will preferably include a translation initiating at thebeginning and a termination codon (e.g., UAA, UGA or UAG) appropriatelypositioned at the end of the mRNA to be translated, with UGA and UAApreferred for mammalian or eucaryotic cell expression.

Expression vectors will preferably include at least one selectablemarker. Such markers include, e.g., dihydrofolate reductase, neomycin,zeocin, hygromycin B or puromycin resistance for eucaryotic cellculture, and kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria or procaryotics. Representative examples ofappropriate hosts include, but are not limited to, bacterial cells, suchas E. coli, Streptomyces, Bacullus subtilis and Salmonella typhimuriumcells; yeast cells such as Saccharomyces cervisiae, Saccharomyces pombeor Pichia pastoris; insect cells such as Drosophila S2, Spodoptera Sf9cells or Sf21 and High Five (BTI-TN-5B1-4) cells; animal cells such as293, 293EBNA, CHO, COS and COS7, BHK, AV12, 3T3, HeLa, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.Vectors preferred for use in bacteria include pET15 and pET30 availablefrom Novagen, pQE70, pQE60 and pQE-9, available from Qiagen; pBSvectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A,pNH46A, available from Stratagene; and ptrc99a, pKR223-3, pKK233-3,pDR540, pRIT5 available from Pharmacia. Preferred eucaryotic vectorsinclude pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene;pSVK3, pBPV, pMSG and pSVL available from Pharmacia, and the commonlyused vector pcDNA3.1 available from Invitrogen. Other suitable vectorswill be readily apparent to the skilled artisan.

Introduction of a vector construct into a host cell can be effected bycalcium phosphate transfection, DRAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16, the entire relevant teachingsof which are incorporated herein by reference.

Another aspect of the present invention relates to fusion proteinscomprising FLINT analogs. For instance, a region of additional aminoacids, for example hexahistidine (His₆) tag , can be added to the aminoor carboxy terminus of a FLINT analog to facilitate purification. Suchregions can be removed prior to final purification of an analog ifdesired. Such methods are described in many standard laboratory manuals,such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel,supra, Chapters 16, 17 and 18, the entire relevant teachings of whichare incorporated herein by reference.

Expression of FLINT Analogs in Host Cells

Using the nucleic acids of the present invention, one may express aFLINT analog in a recombinantly engineered cell, such as bacteria,yeast, insect, or mammalian cells.

Those of skill in the art are knowledgeable in the numerous expressionsystems available for expression of a nucleic acid encoding a FLINTanalog of the present invention. No attempt to describe in detail thevarious methods known for the expression of proteins in procaryotes oreucaryotes will be made.

Briefly, the expression of isolated nucleic acids encoding a FLINTanalog of the present invention will typically be achieved by operablylinking a DNA or cDNA encoding an analog to a promoter (which is eitherconstitutive or inducible), followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither procaryotes or eucaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a promoter to direct transcription, a ribosomebinding site for translational initiation, and atranscription/translation terminator. One of skill would recognize thatmodifications can be made to a protein of the present invention withoutdiminishing its biological activity. Some modifications may be made tofacilitate the cloning, expression, or incorporation of the targetingmolecule into a fusion protein. Such modifications are well known tothose of skill in the art and include, for example, a methionine addedat the amino terminus to provide an initiation site, or additional aminoacids (e.g., poly His) placed on either terminus to create convenientlylocated restriction sites or termination codons or purification handlesequences.

Expression in Procaryotes

Procaryotic cells may be used as hosts for expression of FLINTanalog(s). Procaryotes most frequently are represented by variousstrains of E. coli; however, other microbial strains may also be used.commonly used procaryotic control sequences which are defined herein toinclude promoters for transcription initiation, optionally with anoperator, along with ribosome binding site sequences, include suchcommonly used promoters as the beta lactamase (penicillinase) andlactose (lac) promoter systems (Chang, et al., Nature 198:1056 (1977)),the tryptophan (trp) promoter system (Goeddel, et al., Nucleic AcidsRes. 8:4057 (1980)), T7 phage promoter (Studier, F. W., Methods inEnzymology, 185, 60-89, (1990),and the lambda derived PL promoter andN-gene ribosome binding site (Shimatake, et al., Nature 292:128 (1981)),the teachings of which are incorporated herein by reference. Theinclusion of selection markers in DNA vectors transfected in E. coli isalso useful. Examples of such markers include genes specifyingresistance to ampicillin, tetracycline, chloramphenicol or kanamycin.

The vector is selected to allow introduction into the appropriate hostcell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transformed with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus subtilis and Salmonella (Palva, et al., Gene22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)), theteachings of which are incorporated herein by reference.

Expression in Eucaryotes

A variety of eucaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained below, a nucleic acid encoding a FLINT analog of thepresent invention can be expressed in these eucaryotic systems.

Synthesis of heterologous proteins in yeast is well known. F. Sherman,et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982),the teachings of which are incorporated herein by reference, is awell-recognized work describing the various methods available to producethe protein in yeast. Two widely utilized yeast for production ofeucaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

A FLINT analog of the present invention expressed in yeast can beisolated by standard protein isolation techniques. Monitoring thepurification process can be accomplished by using SDS polyacrylamide gelelectrophoresis (SDS-PAGE), Western blot techniques or radioimmunoassayof other standard immunoassay techniques such as ELISA. can be expressedin these eucaryotic systems.

The nucleic acid sequences encoding FLINT analogs of the presentinvention can also be ligated to various expression vectors for use intransfecting cell cultures of, for instance, mammalian, insect, or plantorigin. Illustrative of cell cultures useful for the production of thepeptides are mamalian cells. Mammalian cell systems often will be in theform of monolayers of cells although mammalian cell suspensions may alsobe used. A number of suitable host cell lines capable of expressingintact proteins have been developed in the art, and include the AV12,HEK293, BHK21, and CHO cell lines. Expression vectors for these cellscan include expression control sequences, such as an origin ofreplication, a promoter (e.g., the CMV promoter, a HSV tk promoter orpgk (phosphoglycerate kinase) promoter), an enhancer (Queen, et al.,Immunol. Rev. 89:49 (1986), the entire teachings of which areincorporated herein by reference), and processing information sites,such as ribosome binding sites, RNA splice sites, polyadenylation sites(e.g., bovine growth hormone poly A addition site), and transcriptionalterminator sequences. Other animal cells useful for production ofproteins of the present invention are available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(8th edition, 1994).

Appropriate vectors for expressing a FLINT analog of the presentinvention in insect cells are usually derived from the SF9 baculovirus.Suitable insect cell lines include mosquito larvae, silkworm, armyworm,moth and Drosophila cell lines such as a Schneider cell line (SeeSchneider, J. Embryol. Exp. Morphol. 27:353-365 (1987), the entireteachings of which are incorporated herein by reference.

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983), the entire teachings of which are incorporatedherein by reference). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors. M. Saveria-Campo,Bovine Papilloma Virus DNA, a Eucaryotic Cloning Vector in DNA CloningVol. II, a Practical Approach, D. M. Glover, Ed., IRL Press, Arlington,Va., pp. 213-238 (1985), the entire teachings of which are incorporatedherein by reference.

Protein Purification

A FLINT analog of the present invention can be recovered and purifiedfrom recombinant cells that express an analog (e.g, E. coli, yeast,insect, or mammalian cell cultures) by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography including immobilized metal ion chelatingpeptide technology, “IMAC,” as taught in U.S. Pat. No. 4,569,974 hereinincorporated by reference, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Analogs of the present invention can be produced by chemical syntheticprocedures, or by recombinant techniques from a procaryotic oreucaryotic host, including bacterial, yeast, higher plant, insect andmammalian cells. Such methods are described in many standard laboratorymanuals, such as Sambrook, supra, Chapters 17.37-17.42; Ausubel, supra,Chapters 10, 12, 13, 16, 18 and 20, the entire relevant teachings ofwhich are incorporated herein by reference. Depending upon the hostemployed, the polypeptides of the present invention may be glycosylatedor non-glycosylated. In addition, polypeptides of the invention can alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

Therapeutic Applications

The apoptosis-triggering molecule, FasL, is an important homeostaticregulator of the immune system, triggering autoreactive peripheral Tcell deletion and dampening the cell-mediated immune response (i.e.activation-induced cell death). FasL also appears to be an importantapoptotic stimulus in non-immune cells under certain conditions (e.g.,inflammation). FasL exists in two forms: membrane-bound and secreted.The latter is derived from proteolytic cleavage and probably plays anadditional role in inflammation by attracting neutrophils. FLINT bindsboth forms of FasL, inhibiting FasL interactions with the membrane-boundFas receptor and with LIGHT, a secreted activator of T cells andpossibly also a trigger for certain cancer cells to die by apoptosis.Constitutive cell surface expression of FasL on certain tissues conveysimmune privilege status, such that cell-mediated immunity does not occuror occurs weakly (due to destruction of invading immune cells bearingFas on their surface). FasL expression is probably regulated in allother tissues; Pas and Pas pathway inhibitors (e.g., FLIP, FAIM) arealso highly regulated.

Following are some of the factors and/or conditions that regulate FasLexpression in humans: inflammation (e.g., acute lung injury),carcinogenesis (e.g., melanoma, colon carcinoma), viral infection (e.g.,Hepatitis C, HIV), autoimmune triggers (e.g., ulcerative colitis,Hashimoto's thyroiditis), and ischemia/re-perfusion (e.g., spinal cordinjury). Undoubtedly many other regulators of FasL expression exist, andit is likely that FasL expression and secretion can be induced in anytissue under certain conditions.

The clinical utility for FLINT analogs is expected to be substantial.Many diseases and/or conditions involving FasL/Fas are potentiallyamenable to therapy with FLINT analog. Examples of suitable diseasesand/or conditions include:

Inflammatory/autoimmune diseases—Rheumatoid arthritis, inflammatorybowel disease, graft-versus-host disease, insulin-dependent diabetes,SIRS/sepsis/MODS, pancreatitis, psoriasis, multiple sclerosis,Hashimoto's thyroiditis, Gravels disease, transplant rejection, SLE,autoimmune gastritis, fibrosing lung disease.

Infectious diseases—HIV-induced lymphopenia, fulminant viral hepatitisB/C, chronic hepatitis/cirrhosis, H. pylori-associated ulceration.

Ischemia/Re-perfusion conditions—Acute coronary syndrome, acutemyocardial infarction, congestive heart failure, atherosclerosis, acutecerebral ischemia/infarction, brain/spinal cord trauma, organpreservation during transplant

Other—Cytoprotection during cancer treatment, adjuvant to chemotherapy,Alzheimer's, chronic glomerulonephritis, osteoporosis, TTP/HUS, aplasticanemia, myelodysplasia. Also of interest are treatment and prevention ofacute lung injury (ALI)/acute respiratory distress syndrome (ARDS);Ulcerative colitis; and Crohn's disease.

FLINT analogs inhibit the binding of FAS to FASL and LIGHT to LTβR andTR2/HVEM receptors, and can be used to treat or prevent a disease and/orcondition that may be associated with such binding.

Runaway apoptosis is one example of a condition caused by excessiveactivation of the PAS/FASL signal transduction pathway that can betreated with the FLINT analogs of the present invention (see U.S.Provisional Application Serial No. 60/112,577, filed Dec. 18, 1998,Kondo et al., Nature Medicine 3(4):409-413 (1997) and Galle et al., J.Exp. Med. 182:1223-1230 (1995), the entire teachings of which areincorporated herein by reference). Runaway apoptosis leads to multiplepathological conditions including organ failure, acute liver failure(e.g., liver failure associated with viral infections affecting theliver, bacterial infections affecting the liver, hepatitis,hepatocellular injury and/or other conditions where hepatocytes undergomassive apoptosis or destruction), kidney failure, and failure ofpancreatic function.

The FLINT analogs of the present invention are generally clinicallyand/or therapeutically useful for diseases which can be treated withFLINT. (See U.S. patent application Ser. No. 09/280,567; and Miwa etal., Nature Medicine 4:1287 (1998), the entire teachings of which areincorporated herein by reference). One example is inflammation caused byFASL induced neutrophil activation. Inflammatory disease associated withneutrophil activation includes sepsis, ARDS, SIRS and MODS.

Other diseases for which FLINT analog is therapeutically useful includeRbeumatoid arthritis (Elliott et al., Lancet 344:1105-10 (1994)),fibroproliferative lung disease, fibrotic lung disease, HIV (Dockrell etal., J. Clin. Invest. 101:2394-2405 (1998)), Ischemia (Sakurai et al.1998 Brain Res 797:23-28), Brain trauma/injury (Ertel et al. 1997 JNeuroimmunol 80:93-6), chronic renal failure (Schelling et al. 1998 LabInvest 78:813-824), Graft-vs-Host Disease (GVHD) (Hattori et al. 1998Blood 11:4051-4055), Cutaneous inflammation (Orteu at al. 1998 J Immunol161:1619-1629), Vascular leak syndrome (Rafi et al. 1998 J Immunol161:3077-3086), Helicobacter pylori infection (Rudi et al. 1998 J ClinInvest 102:1506-1514), Goiter (Tamura et al. 1998 Endocrinology139:3646-3653), Atherosclerosis (Sata and Walsh, 1998 J Clin Invest102:1682-1689), IDDM (Itoh et al. 1997 J Exp Med 186:613-618),Osteoporosis (Jilka et al. 1998 J Bone Min Res 13:793-802), Crohn'sDisease (van Dullemen et al. 1995 Gastroenterology 109:129-35), organpreservation and transplant (graft) rejection (Lau et al. 1996 Science273:109-112), Sepsis (Faist and Kim. 1998 New Horizons 6:S97-102),Pancreatitis (Neoptolemos et al. 1998 Gut 42:886-91), Cancer (melanoma,colon and esophageal) (Bennett et al. 1998 J Immunol 160:5669-5675),Autoimmune disease (IBD, psoriasis, Down's Syndrome (Seidi et al.,Neuroscience Lett. 260:9 (1999) and multiple sclerosis (D'Souza et al.1996 J Exp Med 184:2361-70).

Co-pending U.S. patent application, Ser. No. 09/280567, entitled“Therapeutic Applications of mFLINT Polypeptides,” filed Mar. 30, 1999,herein incorporated by reference, discloses other diseases which can betreated with FLINT analog. Examples include Alzheimer's Disease;End-stage renal disease (ESRD); mononulceosis; EBV; Herpes; antibodydependent sytotoxicity; hemolytic and hypercoagulation disorders such asvascular bleeds, DIC (disseminated intervascular coagulation),eclampsia, HELLP (preeclampsia complicated by thrombocytopenia,hemolysis and disturbed liver function), HITS (heparin inducedthromobcytopenia), HUS (hemolytic uremic syndrome), and preeclampsia;hematopoeitic disorders such as aplastic anemia, thrombocytopenia (TTP)and myelodysplasia; and hemolytic fever caused, for example, by E.bola.

In the case of organ preservation in preparation for harvesting, forinstance, FLINT analog is useful prophylactically to prevent theapoptosis associated with ischemia reperfusion injury to the organ onceit is removed from the donor. In one embodiment of this aspect of theinvention, FLINT analog is administered to the organ donor prior toharvesting the organ. After harvesting the organ, FLINT analog is addedto a suitable medium for transport/storage of the organ. Alternatively,the harvested organ is perfused with a medium containing FLINT analogprior to transplantation into a recipient. Suitable media for thispurpose are known, for example, the media disclosed in EP 0356367 A2,herein incorporated by reference. The method may also include treatingthe transplant recipient with FLINT analog prior to and/or after thetransplant surgery.

A typical method involves pre-treating the organ donor with an effectiveamount of FLINT analog prior to organ harvesting. Alternatively, orconjunctively, the harvested organ may be perfused or bathed in a FLINTanalog-containing solution. This method may be employed, for example,with kidney, heart, lung and other organs and tissues.

The ability of FLINT analogs to retard apoptosis under ischemicconditions is useful for preserving organs and tissues harvested fortransplantation. Ischemic conditions include, but are not limited to,neuronal ischemia, limb crush injuries, spinal cord injuries, myocardialinfarct including acute, subacute and chronic sequelae and relatedclinical syndromes, congestive heart failure. Also, innocent bystandertissues which are damaged during chemotherapy, radiation therapy, toxicdrugs, trauma, surgery and other stresses can be treated with FLINTanalog. One example of such a disease is mucositis which can be alife-threatening side-effect of cancer treatment.

In another embodiment, therefore, the invention relates to administeringFLINT analog to an organ donor prior to harvesting an organ. After organharvesting, FLINT analog is added to a suitable medium for transportand/or storage of the organ. Alternatively, the harvested organ isperfused with a medium containing FLINT analog prior to transplantationinto a recipient. Suitable media for perfusion are known, for example,the medium disclosed in EP 0356367 A2. The method may also includetreating the transplant recipient with FLINT analog prior to and/orafter the transplant surgery.

In this aspect, an organ donor is pre-treated with an effective amountof FLINT analog prior to organ harvesting. Alternatively, orconjunctively, the harvested organ may be perfused or bathed in asolution containing FLINT analog. The method may be employed, forexample, with kidney, heart, lung and other organs and tissues.

Acute Lung Injury and Acute Respiratory Distress Syndrome

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)represent disease entities that differ only in the severity of thehypoxemia present at diagnosis. A widely accepted parameter fordiagnosis is the PaO2 to FiO2 ratio, according to which ARDS patientsmanifest a ratio of less than or equal to 200 mm Hg, whereas ALIpatients exhibit values of less than or equal to 300 mm Hg. ARDSrepresents a more severe form of ALI. Numerous mediators are likely tocontribute to the pathogenesis of ARDS/ALI with neutrophils playing aprominent role. While multiple precipitating factors are probable in thedevelopment of ARDS, both direct and indirect, the major cause appearsto be sepsis and the systemic inflammatory response syndrome, accountingfor approximately 40% of cases. Mortality in ARDS is high approximating40%, with most deaths occurring within the first 2 to 3 weeks. There isno currently available, approved pharmacologic therapy for ARDS andtreatment at present is limited to aggressive supportive care.

There is evidence that ARDS may be mediated by soluble FasL/Pasinteraction in humans (Matute-Bello et al., J. Immunol. 163, 2217-2225,1999). FLINT analog, by binding to FasL, could inhibit FasL-mediatedapoptosis of pneumocytes and/or endothelial cells, thus inhibiting orpreventing the progression from acute inflammatory insult to ALI, andfrom ALI to ARDS.

Therefore, in another embodiment, the present invention relates to theuse of FLINT analog to inhibit and/or treat ALI and/or ARDS comprisingthe administration of a therapeutically effective amount of FLINT analogto a person in need thereof.

Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is the fourth leading causeof non-accidental death in the United States following heart disease,cancer and cerebral vascular disease. COPD is an obstructive airwaydisorder encompassing multiple conditions including chronic bronchitis,emphysema, bronchiectasis, and chronic asthma. COPD is slowlyprogressive and produces an irreversible decline in lung function.Chronic hypoxemia and hypercapnia are the eventual outcomes of thedisorder. The mechanism by which COPD disrupts lung function appears toinvolve dysregulated apoptosis. Plasma samples from patients sufferingfrom COPD exhibit higher concentrations of soluble Fas compared withhealthy control subjects (See Yasuda et al. Resp. Med. 92, 993-999,1998). The increased levels of soluble Fas in COPD patients may reflectincreased Fas-induced apoptosis.

In another embodiment, the present invention relates to the use of FLINTanalog to treat and/or inhibit COPD in a patient in need thereof byadministering a therapeutically effective amount of FLINT analog.

Pulmonary Fibrosis (PF)

Pulmonary fibrosis (also known as fibrosing lung disease) occurs as anend result of the process of attempted healing during acute or chroniclung injury. The pathological mechanism of such lung injury can be anyof various factors that first trigger an inflammatory response in thealveoli or surrounding interstitium and subsequently triggeralveolar/interstitial fibrosis (i.e. the repair response). Fibrosis inother tissues such as the epidermis or the peritoneum, leads to visiblescarring or adhesions, respectively. Pulmonary fibrosis, in contrast,leads to restrictive lung disease (decreased lung capacities anddecreaased oxygen diffusion). Conditions associated with pulmonaryfibrosis include but are not limited to: idiopathic pulmonary fibrosis,connective tissue diseases (e.g. lupus, scleroderma), drug-induced lungdisease (e.g. bleomycin), pneumoconioses (e.g. asbestosis), sarcoidosis,eosinophilic granulomatosis, hypersensitivity pneumonitis, and otherdiseases asscoiated with severe lung inflammation that can result inacute lung injury and/or acute respiratory distress syndrome (e.g.trauma, sepsis, near-drowning, gastric aspiration, shock, etc.).Fibrosis of the airways is also a feature of the chronic inflammation inCOPD.

The etiology of PF may involve FasL/Fas-triggered apoptosis. Indeed, anintact FasLiFas system is essential in the etiology of bleomycin-inducedPF in mice (See Kuwano K. et al. J. Clin. Invest. 104, 13-19 (1999).

In another embodiment the present invention relates to the use of FLINTanalog to inhibit and/or treat PF. For example, FLINT analog can beadministered acutely at the time of an inflammatory insult to the lung(e.g. during bleomycin treatment) to prevent PF from occurring.

A “subject” is a mammal in need of treatment, preferably a human, butcan also be an animal in need of veterinary treatment, e.g., domesticanimals (e.g., dogs, cats, and the like), farm animals (e.g., cows,sheep, pigs, horses, and the like) and laboratory animals (e.g., rats,mice, guinea pigs, and the like).

An “effective amount” of a FLINT analog is an amount which results in asufficient inhibition of one or more processes mediated by the bindingof FAS to FAS Ligand or LIGHT to LTβR and/or TR2/HVEM so as to achieve adesired therapeutic or prophylactic effect in a subject with a diseaseor condition associated with aberrant FAS/FAS Ligand binding and/orLIGHT mediated binding. One example of such a process is runawayapoptosis. Alternatively, an “effective amount” of a FLINT analog is aquantity sufficient to achieve a desired therapeutic and/or prophylacticeffect in a subject with inflammation caused by FAS Ligand inducedneutrophil activation or any of the other aforementioned diseasesassociated with aberrant FAS Ligand activity.

A “desired therapeutic and/or prophylactic effect” in a subject with adisease or condition includes the amelioration of symptoms, or delay inonset of symptoms, associated with such disease. Alternatively, a“desired therapeutic and/or prophylactic effect” includes an increasedsurvival rate or increased longevity for the subject with the disease.

The amount of FLINT analog administered to the individual will depend onthe type and severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. It will also depend on the degree, severity and type ofdisease. The skilled artisan will be able to determine appropriatedosages depending on these and other factors.

As a general proposition, the total pharmaceutically effective amount ofthe FLINT analogs of the present invention administered parenterally perdose will be in the range of about 1 μg/kg/day to 10 mg/kg/day ofpatient body weight, particularly 2 mg/kg/day to 8 mg/kg/day, moreparticularly 2 mg/kg/day to 4 mg/kg/day, even more particularly 2.2mg/kg/day to 3.3 mg/kg/day, and finally 2.5 mg/kg/day, although, asnoted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day. If given continuously,the FLINT analogs of the present invention are typically administered ata dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4injections per day or by continuous subcutaneous infusions, for example,using a mini-pump. An intravenous bag solution may also be employed. Thelength of treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Pharmaceutical compositions containing the FLINT analogs of the presentinvention may be administered orally, rectally, intracranially,parenterally, intracisternally, intravaginally, intraperitoneally,topically (as by powders, ointments, drops or transdermal patch),transdermally, intrathecally, bucally, or as an oral or nasal spray. By“pharmaceutically acceptable carrier” is meant a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The term “parenteral” as used hereinincludes, but is not limited to, modes of administration which includeintravenous, intramuscular, intraperitoneal, intrasternal, subcutaneousand intraarticular injection, infusion and implants comprising FLINTanalogs.

The FLINT analogs of the present invention are also suitablyadministered by sustained-release systems. Suitable examples ofsustained-release compositions include semi-permeable polymer matricesin the form of shaped articles, e.g., films, or microcapsules.Sustained-release matrices include polylactides (U.S. Pat. No.3,773.919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R.Langer et al., J. Biomed.Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105(1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988). Other sustained-releasecompositions also include liposomally entrapped FLINT analog. Suchliposomes are prepared by methods known per se: DE 3,218,121; Epstein etal., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EDP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimal TNFRpolypeptide therapy.

For parenteral administration, the FLINT analogs of the presentinvention are formulated generally by mixing at the desired degree ofpurity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation. For example,the formulation preferably does not include oxidizing agents and othercompounds that are known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting the FLINT analogsof the present invention uniformly and intimately with liquid carriersor finely divided solid carriers or both. Then, if necessary, theproduct is shaped into the desired formulation. Preferably the carrieris a parenteral carrier, more preferably a solution that is isotonicwith the blood of the recipient. Examples of such carrier vehiclesinclude water, saline, Ringer's solution, and dextrose solution.Non-aqueous vehicles such as fixed oils and ethyl oleate are also usefulherein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The FLINT analogs of the present invention are typically formulated insuch vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml,preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understoodthat the use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of salts of the FLINT analogsof the present invention.

Polypeptides to be used for therapeutic administration must be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticpolypeptide compositions generally are placed into a container having asterile access port. for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

FLINT analogs ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous solution of one of the FLINTanalogs of the present invention, and the resulting mixture islyophilized. The infusion solution is prepared by reconstituting thelyophilized polypeptide using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainers) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, theFLINT analogs of the present invention may be employed in conjunctionwith other therapeutical compounds.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

EXAMPLE 1 Production of a Vector for Expressing FLINT Analog R218Q

FLINT variant R218Q was constructed by mutagenic PCR starting from awild-type FLINT template. See e.g. Saiki R. K. et al. Science239:487-491 (1988), and “Current Protocols in Molecular Biology”, Vol 1,section 8.5.7 (John Wiley and Sons, Inc. publishers) the entire contentsof which are herein incorporated by reference. The R218Q mutantsubstitutes an arginine residue found at amino acid 218 with glutamine.

The mutagenic PCR process utilized a SOEing reaction (i.e. StrandOverlap Extension) to create specific mutations in the native FLINTtemplate for the purpose of changing the amino acid sequence at position218, and further for introducing restriction enzyme tags foridentification purposes.

Generally, SOEing reactions require the use of four primers, two in theforward orientation (termed A, SEQ ID NO:5, and C, SEQ ID NO:7) and twoin the reverse orientation (termed B, SEQ ID NO:6 and D, SEQ ID NO:8).The SOEing reaction amplifies a nucleic acid sequence (e.g. genesequence) in two stages. The first step is to amplify “half” the gene byperforming an A to B reaction followed by a separate C to D reaction. Inconstructing the R218Q mutant, the B and C primers were targeted to thesame area of the gene but on opposite strands. Mismatch priming fromboth oligonucleotide primers institutes the mutation. After these tworeactions were completed, the products were isolated and mixed for useas the template for the A to D reaction, which yields the desiredmutated product.

The primers involved in the cloning of R218Q were:

Primer A:  CF 107   (39 nt) GCACCAGGGTACCAGGAGCTGAGGAGTGTGAGCGTGCCGPrimer B:  CF 111   (44 nt) TCAGCTGCAAGGCGGCGCGCCCCGCTTGTGGTGTCGGACCCCAGPrimer C:  CF 112   (44 nt) GGGGTCCGACACCACAAGCGGGGCGCGCCGCCTTGCAGCTGAAGPrimer D:  CF 110   (43 nt) GCACAGAATTCATCAGTGCACAGGGAGGAAGCGCTCACGGACG

The nucleotides shown in bold represent changes made to the wild typesequence. Using the forward primer C as a reference, the bold G and Cshow the silent changes necessary to introduced an AscI site. Thisrecognition site is underlined in primers B and C. The bold CAA showsthe amino acid substitution of glutamine (CAA) for arginine (AGG).

The 311 base pair amplified fragment carrying the R218Q mutation wassub-cloned using a 51 KpnI site (GGTACC) and a 3′ EcoRI site (GAATTC).The native FLINT sequence has a naturally occurring internal KpnI sitearound amino acid position 176. The EcoRI site was introduced forsub-cloning purposes and lies downstream of the stop codons. These sitesare underlined in primers A and D respectively. The 311 bp fragment wasincorporated into the full length FLINT sequence. This was accomplishedthrough the following steps:

I. The 311 bp fragment was placed into an intermediate vector,pCR2.1-TOPO, which utilizes the adenine overhangs established after PCRfor ligation.

II. Once incorporated, a KpnI to EcoRI digestion removed a 289 bpfragment. (Note: the size of the PCR fragment decreased from 311 bp to289 bp due to the digestion). The mutated fragment was used to replacethe corresponding segment in the wild type FLINT gene by directionalligation.

III. FLINT/pJB03 was digested with KpnI to EcoRI to produce twofragments

Fragment 1: 6070 bp

Fragment 2: 289 bp

IV. The 6070 bp fragment carrying the FLINT gene was isolated andligated with the 289 bp PCR product removed from the pCR 2.1-TOPO vectorto create R218Q/pJB03. Positive clones were identified by restrictiondigestion and subsequently confirmed by sequence analysis.

The R218Q analog contained in R218Q/pJB03 was shuffled into the pIG3vector by means of a NheI to XbaI undirected ligation. R218Q/pJB03 wasdigested with NheI to XbaI to yield fragments of 932 and 5427 bp. The932 bp FLINT R218Q containing fragment was isolated. The 932 bp NheI andXbaI fragment of R218Q was ligated with the 8510 bp linearized pIG 3vector to generate clones in both forward and inverse orientations.

EXAMPLE 2 Procaryotic Expression and Purification of Recombinant FLINTAnalogs

An expression vector that carries an open reading frame (ORF) encodingFLINT analog R218Q is transformed into competent E. coli strain BL21(DE3)(hsdS gal cIts857 ind1Sam7nin5lacUV5-T7gene 1)available fromNovagen (Madison Wis.) using standard methods. Multiple transformants,selected for resistance to kanamycin, were chosen at random and used toinoculate LB/kanamycin media. Cultures were grown at 37° C. with shakingto cell density of OD₆₀₀=0.8-1.0 at which point FLINT protein synthesiswas induced with 1 mM IPTG. Cultures were grown at 37° C. for 3hpostinduction before the cells are harvested. FLINT analog productencoded by the vector-borne ORF was purified from the insoluble fractionof cell lysate, the inclusion bodies. In brief, the purificationconsisted of inclusion bodies preparation and solubilization, andprotein refolding followed by cation-exchange and size-exclusionchromatography procedures, respectively.

EXAMPLE 3 Construction of Vector pIG3 for Expression of FLINT Analogs inMammalian Cells

A bicistronic expression vector is constructed by insertion into themammalian expression vector PGTD (Gerlitz, B. et al., 1993, BiochemicalJournal 295:131) a PCR fragment encoding an “internal ribosome entrysite”/enhanced green fluorescent polypeptide (IRES/eGFP). This newvector, designated pIG3, contains the following sequence landmarks: theEla-responsive GBMT promoter (D. T. Berg et al., 1993 BioTechniques14:972; D.T. Berg et al., 1992 Nucleic Acids Research 20:5485); amultiple cloning site (MCS); the IRES sequence from encephalomyocarditisvirus (EMCV); the eGFP coding sequence (Cormack, et al., 1996 Gene173:33, Clontech); the SV40 small “t” antigen splicesite/poly-adenylation sequences; the SV40 early promoter and origin ofreplication; the murine dihydrofolate reductase (dhfr) coding sequence;and the ampicillin. resistance gene and origin of replication frompBR322.

Based on the human FLINT cDNA sequence, the forward and reverse PCRprimers are synthesized bearing BclI restriction sites at theirrespective 5′ends. These primers are used to PCR amplify the FLINTanalog CDNA. The human FLINT cDNA orientation and nucleotide sequenceare confirmed by restriction digest and double stranded sequencing ofthe insert. The approximately 900 base pair amplified FLINT analog PCRproduct was digested with restriction endonucleases NheI and XbaI,respectively, to generate a fragment bearing NheI and XbaI sticky ends.This fragment was subsequently ligated into a unique XbaI site of pIG3to generate recombinant plasmid pIG3-FLINT. The insert encoding theanalog can be modified at the C-terminus of the analog to introduce acleavable hexahistidine (His6) cassette to facilitate analogpurification.

EXAMPLE 4 Isolation of High-Producing FLINT Analog Clone from AV12 RGT18Transfectants

The recombinant plasmid carrying the FLINT gene encodes resistance tomethotrexate. In addition, the construct contains a gene encoding afluorescent polypeptide, GFP, on the same transcript and immediately 3′to the FLINT gene. Since high level expression of GFP would require ahigh level of expression of the FLINT-GFP mRNA, highly fluorescentclones would have a greater probability of producing high levels ofFLINT.

AV12 RGT18 cells are transfected using a calcium phosphate procedurewith recombinant pIG1 plasmids containing FLINT analogs. Cells resistantto 250 nM methotrexate are selected and pooled. The pool of resistantclones is subjected to fluorescence assisted cell sorting (FACS), andcells having fluorescence values in the top 5% of the population aresorted into a pool and as single cells. High fluorescence pools aresubjected to two successive sorting cycles. Pools and individual clonesfrom the first and second cycles are analyzed for FLINT analogproduction by ELISA. Pools or clones expressing FLINT analog at thehighest level are used for scale-up and FLINT analog purification.

EXAMPLE 5 Quantitation of FLINT Analogs

FLINT analogs can be quantitated in crude media of transfected cells andduring purification procedure by developed FLINT ELISA. ELISA usesanti-FLINT polyclonal antibody TKD-028(1494) as a capture antibody andbiotinylated anti-FLINT TKD-076A as a primary antibody in a “sandwich”assay. ELISA is developed by streptavidin derivatized horse radishperoxidase (SA-HRP) using OPD as a substrate and monitoring theabsorbance at 490 mn. The useful range of such an ELISA is from 0.2-20ng/ml.

EXAMPLE 6 FLINT Analogs Inhibit FasL Induced Jurkat Cell Apoptosis

A bioassay measuring the prevention of apoptosis (i.e. cell survival)was performed using FLINT and a variety of FLINT analogs produced inmammalian cell culture. For this purpose, 25 μl of Jurkat cells (5×10⁴cells/well) were added to each well of a 96-well plate and mixed with 25μl of recombinant human FasL (final concentration 150 ng/ml), and either50 μl of FLINT or FLINT analog. serial dilutions ranging from 0 to 1mg/ml were tested in the assay. Cells were incubated at 37° C. overnight. Twenty μl of MTS tetrazolium compound (Promega Corporation,Madison, Wis.) was added to each well and the incubation carried out for2h at 37° C. Absorbance at 490 nm was recorded using a plate reader.

The results are summarized in the Table below and in FIGS. 2-4. Analogsthat changed arginine at position 218 to glutamine, glutamic acid,alanine, glycine, or valine showed activity in this assay. Bioactivitywas not a function of the cell type from which the sample was prepared.For example, R218Q purified from AV12 cells (FIG. 2), or from 293 EDNAcells (FIG. 3) were active in the assay.

Other analogs were also tested. For example, a double mutant analog thatreplaced threonine at position 216 with proline, and arginine atposition 218 with glutamine, was active in the assay. Also active wasthe multi-substituted analog [R34N, D36T, D194N, S196T, R218Q] analogwas active in this assay (FIG. 4).

INHIBITION OF JURKAT CELL FLINT/FLINT ANALOG APOPTOSIS FLINT ++ R218Q ++R218A + R218G + R218V + P217Y + R34N, D36T, R218Q + R218E ++ R34N, D36T,D194N, S196T, + R218Q T216P, R218Q ++

EXAMPLE 7 Testing FLINT Analogs for Inhibition of Apoptosis of JurkatCells Induced by FasL

A bioassay measuring the prevention of apoptosis (i.e. cell survival) isperformed using FLINT and a variety of FLINT analogs (see Table below)produced in mammalian cell culture. FLINT analogs to be tested include:G214(1), G214(2), G214(3), G214(4), G214(5); P215(1), P215(2), P215(3),P215(4), P215(5); T216(1), T216(2), T216(3), T216(4), T216(5); P217(1),P217(2), P217(3), P217(4), P217(5); R218(1), R218(2), R218(3), R218(4),R218(5); A219(1), A219(2), A219(3), A219(4), A219(5); G220(1), G220(2),G220(3), G220(4), G220(5); R221(1), R221(2), R221(3), R221(4), R221(5);A222(1), A222(2), A222(3), A22(4), A222(5). Parenthetical numbersdesignate specific amino acid subgroups as follows with the proviso thatthe replacement residue not be the same as that in native FLINT:

(1) any of the natural 20 amino acids;

(2) Asp or Glu

(3) His, Arg, or Lys

(4) Cys, Thr, Ser. Gly, Asn, Gln, Tyr

(5) Ala, Pro, Met, Leu, Ile, Val, Phe, Trp

For this purpose, 25 μl of Jurkat cells (5×10⁴ cells/well) are added toeach well of a 96-well plate and mixed with 25 μl of recombinant humanFasL (final concentration 150ng/ml), and either 50 μl of FLINT or aFLINT analog. Serial dilutions ranging from 0 to 1 mg/ml are tested inthe assay. Cells are incubated at 37° C. over night. Twenty μl of MTStetrazolium compound (Promega Corporation, Madison, Wis.) is added toeach well and the incubation carried out for 2h at 37° C. Absorbance at490 nm is recorded using a plate reader.

TESTING REPRESENTATIVE FLINT ANALOGS FOR BIOLOGICAL ACTIVITY G214D,G214H, G214T, G214A P215D, P215H, P215T, P215A T216D, T216H, T216S,T216A P217D, P217H, P217T, P217A A219D, A219H, A219T, A219M G220D,G220H, G220T, G220A R221D, R221H, R221T, R221A A222D, A222H, A222T,A222M

EXAMPLE 8 Large Scale FLINT Analog Polypeptide Purification

Large scale production of FLINT analog (containing a 6 histidine tag) isperformed by growing stable pools in roller bottles. After reachingconfluency, cells are further incubated in serum-free medium for 5 to 7days to secrete maximum amounts of FLINT analog into the medium. Mediumthat contains FLINT analog is concentrated in an Amicon ProFlux M12tangential filtration system to 350 ml. The concentrated medium ispassed over an IMAC column (Immobilized Metal-AffinityChromatography(Pharmacia, 5 to 10 ml column) at a flow rate of 1 ml/min.The column is washed with buffer A(PBS, 0.5 M NaCl, pH 7.4) until theabsorbency (280 nm) returned to baseline and the bound polypeptide iseluted with a linear gradient from 0.025 M-0.5 M Imidazol(in buffer A)developed over 60 min. Fractions containing the FLINT analog are pooled.This material is passed over a Benzamidine Sepharose column(1 to 5 ml)to remove the thrombin at a flow rate of 1 ml/min. The columnflow-through containing the FLINT analog is concentrated using anUltrafree centrifugal filter unit (Millipore) to 2 ml. This material ispassed over a 16/60 Superdex 200 sizing column (Pharmacia)equilibratedwith PBS, 0.5 M NaCl, pH 7.4. Fractions containing the FLINT analog areanalyzed by SDS-PAGE, and the N-terminal sequence of the purifiedpolypeptide confirmed to be FLINT.

EXAMPLE 9 Biophysical Characterization of Purified FLINT Analogs

The structural integrity and physical and chemical stability of FLINTanalogs are characterized as follows.

Structural analysis of proteins includes assessment of secondarystructure normally obtained by far-UV CD analysis. Usually 100 μl of 1mg/ml FLINT analog in phosphate buffer, pH 7.4 is used to scan from240-180 nm in 0.5 nm steps, 1 nm bandwidth, with 3 sec time constant, anaverage of 3 scans in a 0.01 cm cell at room temperature. Near-UV CDspectra reporting information on the tertiary structure is taken from240-350 nm, 0.5 nm step, 1 nm bandwidth, 5 second time constant, withaverage of 3 scans at room temperature.

Intrinsic tryptophan fluorescence is measured with the followingparameters: excitation through a 1 cm pathlength cell at 298 nm with a2/2 nm slit width with emission collected from 305-400 nm with a 2/2 nmslit width, 05 nm step, through a 0.4 cm pathlength with 1 secintegration time. A “blue shift” is generally indicative that aromaticresidues are more deeply buried in the protein structure and is oftenaccompanied by improved pharmaceutical properties.

Quaternary structure of FLINT analogs can be examined by equilibriumsedimentation analysis performed in an ultracentrifuge with 3 mm widthcells. Analogs with similar equilibrium sedimentation values compared tonative FLINT are preferred.

Physical stability analysis includes examination of the propensity foraggregation of FLINT analogs as a reflection of their surfaceproperties. Physical stability assays are described in the followingparagraphs.

Dynamic Light Scattering Assay(DLS): A FLINT analog solution is dilutedinto either a) PBS, pH 7.4 or b) PBS, 0.5 M NaCl or c) PBS, pH 7.4 and 3mg/mL m-Cresol, containing 0.1 to 5 mg/mL protein. The pH is adjusted to7.4 (±0.05) with HCl/NaOH and filtered into a 6×50 mm borasilica type-Iglass tube. The average light-scatter intensity weighted particle sizeis collected on a Brookhaven BI900 Instrument consisting of a goniometerat a 90° angle, digital correlator, and a Lexel model 3500 argon ionlaser adjusted to the 488-nm line. The experimentally determinedautocorrelation function C(t) is analyzed by the cumulants method toyield the hydrodynamic diameter. The time before a significant change inparticle size, or lag time, is determined by fitting linear lines to thepre-growth and growth phase data points. The intersection is defined asthe lag time. Decreased light scattering by an analog compared withnative FLINT at the same concentration and temperature is generallyindicative that the analog aggregates to a lesser extent than nativeprotein.

Differential Scanning Callorimetry (DSC): Physical stability is alsoreflected in the melting temperature (Tm) of the protein by DSC(Differential Scanning Callorimetry). Usually FLINT analogs are scannedfrom 5°-100° C. with a 60° C./h scan rate and a 16 second filteringparameter. Higher melting temperatures are generally indicative ofphysical stability.

Chemical stability of FLINT analogs diluted to 0.5 mg/ml is monitored byanalyses by reversed-phase HPLC (RP-HPLC) and size exclusionchromatography. The reversed-phase method consists of anacetonitrile/TFA gradient systems optimized for FLINT with detection at214 nm using a Zorbax 300SB-C8 column at 40° C. The size exclusionmethod consists of a PBS mobile phase at pH 7.4 on a Superdex-75(3.2×300mm) column at room temperature. Changes in the reverse-phasechromatogram are generally indicative of chemical instability.

EXAMPLE 10 In vivo Testing of FLINT Analogs for Treatment of LiverDamage

Liver damage was induced in vivo in a mouse model using the method ofTsuji H., et al, 1997, Infection and Immunity, 65(5):1892-1898. Micewere challenged with a low dose of lipopolysaccaride (LPS) to induceacute and massive hepatic injury. The ability of FLINT and FLINT analogsto protect against acute inflammation and apoptosis was determined.Briefly, BALB/c mice (Harlan) were given intravenous injections (thelateral tail vein) of 6 mg of D(+)-Galactosamine (Sigma, 39F-0539) in100 μl of PBS (GIBCO-BRL) and 3 μg of Lipopolysaccharide B E.coli 026:B6(LPS) (Difco, 3920-25-2) in 100 μl of PBS. After LPS challenge, theanimals were injected intravenously with FLINT (1-200 μg) or a FLINTanalog (1-200 μg), at 4 hour after LPS treatment. The survival rates ofthe mice were determined after 48 hours (see FIG. 7).

A correlation was observed in the percent survival of animals and theamount of FLINT or analog administered. In one study, involving 5animals per test group, analogs R218Q, and [R34N, D36T, D194N, S196T,R218Q] protected animals from liver damage in a dose dependent fashion(see FIG. 7). In a second experiment, involving 10 animals per testgroup, 7 of 10 animals survived at 20 hours post-treatment with FLINT,R218Q, and [R34N, D36T, D194N, S196T, R218Q]. At 24 hourspost-treatment, 7 of 10 animals survived with FLINT treatment, and 6 of10 survived with R218Q or [R34N, D36T, D194N, S196T, R218Q].

EXAMPLE 11 FLINT Analog/FAS Ligand Binding Assay

The binding between FLINT analogs and Fas Ligand can be confirmed andthe binding properties determined (e.g., kinetics, specificity,affinity, cooperativity, relative binding pattern) using real-timebiomolecular interaction analysis (hereinafter BIA). To monitorbiomolecular interactions, BIA uses optical phenomenon surface plasmonresonance. Detection of binding interactions depends on changes in themass concentration of macromolecules at the biospecific interface. Thefollowing materials and methods were used.

Biacore® 2000 device (Biacore AB, Rapsgatan 7, S-754 50 Uppsala, Sweden)

Sensor Chip CM5 (Biacore)

Amine coupling kit (Biacore)

Washing buffer: HBS-EP (Biacore)

Guanidine Isothiocyanate Solution (GibcoBRL)

Fas Ligand (Kamiya Biomedical Company, 910 Industry Drive, Seattle,Wash. 98188)

FLINT Analogs

Pas/Fc chimera (R&D Systems)

Immobilization Protocol:

FasL or the FLINT analog are immobilized through their primary aminegroup on lysine residues onto carboxymethyldextran polymer attached to agold surface (Sensor Chip CM5). Immobilization is carried out using theamine coupling kit (Biacore) according to the manufacturer's protocol.Briefly, 100 μl of either FasL or FLINT analog solutions (10-25 μg/ml insodium acetate buffer 20 mM, pH 5.0) is loaded onto an activated CM5chip. After coupling, excess reactive groups on the surface aredeactivated with 1M ethanolamine hydrochloride pH 8.5. The chip is thenwashed with a sodium acetate buffer 20 mM, pH 5.0 to removenon-covalently bound material.

Interaction Analysis:

To analyze the interaction between FasL and FLINT analogs, solutionscontaining the FLINT analogs are passed over the chip with PasLimmobilized thereon. The amount of the FLINT analog associated with FasLis determined by measuring the surface plasmon resonance signal (inresponce units, RU). Typically, FLINT analog solutions at differentconcentrations in HBS-ET buffer are loaded on the sensor chip for 10minutes at a flow rate of 5 μl/min. The chip is then washed with HBS-ETbuffer (10 mM Hepes, pH 7.4; 150 mM NaCl, 3 mM EDTA, 0.005% SurfactantP20) for 2 minutes at a flow rate of 5 μl/min. The bioactive surface ofthe chip is then regenerated by exposure to 0.8 M guanidineisothiocyanate for 2 minutes at a flow rate of 5 μl/min and then HBS-EPbuffer for 2 minutes at 5 μl/min flow rate.

The converse reaction can also be done whereby a FasL solution is passedover a chip having FLINT analog immobilized thereon. The amount of FasLassociated with the FLINT analog is determined using surface plasmonresonance signal (in response units, RU).

Determination of Affinity Constants:

The data is evaluated and binding parameters determined using BIAevaluation 3.0.2 software (Biacore). The Kd for the interaction betweenFLINT and FasL and between FasL and Fas was determined using theprotocols described above. The results are shown below:

Interacting molecules:

Immobilized—in solution

Kd (nM)

FasLigand (monomer)—FLINT

1.13×10⁻⁷

FasLigand (monomer)—Fas

1.62×10⁻⁷

FLINT analog—FasLigand (trimer)

0.63×10³¹ ⁹

EXAMPLE 12 Analytical Thrombin Digestion of FLINT and FLINT Analogs

In separate reaction tubes, 1 ug of FLINT and 1 ug of the FLINT analogsR218Q , ER34N, D36T), and [R34N, D36T, D194N, S196T, R218Q] wereincubated in a buffer containing 20 mM Tris, 150 mM NaCl, pH 7.4 or PBS,0.5 M NaCl, 10% glycerol and thrombin at a weight ratio of 1 to 100(thrombin to FLINT/analog). The reaction mixtures were incubated forvarying times at either 25 ° C. or 37° C. Aliquots from the reactionmixtures were analyzed by SDS-PAGE to detect cleavage at position 218.The results are presented in FIG. 1. As expected, FLINT was digested bythrombin to produce the FLINT metabolite. For example, by 50 min. almosthalf the FLINT and a control analog [R34N, D36T] were proteolized and at4 hr. >90% was proteolized. In contrast, proteolysis at the 218 positionwas not detected on samples of the R218Q and [R34N, D36T, D194N, S196T,R218Q] analogs even out to the 4 hour time point (FIG. 1).

EXAMPLE 13 Metabolic Stability In vitro of FLINT Analog R218Q

FLINT derived from AV12 cells was supplied as a solution of 0.16 mg/mlin phosphate buffered saline/0.5 M NaCl/10% glycerol. FLINTanalog(R218Q), derived from 293 EBNA cells, was supplied as a solutionof 0.12 mg/ml in phosphate buffered saline/0.5 M NaCl/10% glycerol.Materials were stored at 4° C. until use. FLINT and FLINT analog (R218Q)were radiolabeled with ¹²⁵I-NaI using the IODO-BEADS iodination reagent(PIERCE). Radiolabeled test articles were >90% precipitable intrichloroacetic acid. Radiolabeled proteins were stored at −20° C. untiluse.

For in vitro analysis, mouse blood samples were collected by cardiacpuncture into clotting tubes (serum tubes, no anti-coagulant) from maleICR mice (weighing 35 g to 45 g). Immediately thereafter, ¹²⁵I-FLINT or¹²⁵I-FLINT analog (R218Q) was added to the blood collection tubes. Thetubes were then placed in a water bath at 37° C. and allowed to clot for1 hr. Serum was prepared by sedimentation and assayed by reversed-phaseHPLC.

Serum or plasma samples were applied directly to a Vydac C4 Column(4.6×250 MM). Compound(s) were eluted from the column with a lineargradient of 15-55% B in 40 min at 1 ml/min. Solvent A=H₂O/0.1% TFA;solvent B=acetonitrile/0.08% TFA. Eluate was monitored at 214 nm.Fractions of column eluate (1 ml) were collected. Radioactivity infractions was analyzed directly by gamma counting.

In vitro incubation. After a 1 h incubation with ICR mouse blood,approximately 75% of ¹²⁵I-FLINT was degraded to a product having theretention characteristics of the FLINT metabolite, (i.e. residues 1-218of SEQ ID NO:1). (FIG. 5A) In contrast, the FLINT analog (R218Q) wascompletely resistant to proteolytic degradation (FIG. 5B).

EXAMPLE 14 Metabolic Stability in vivo of FLINT Analog R218Q

Studies designed to test the stability and resistance of FLINT analog.to proteolytic digestion in vivo were performed in male ICR mice (bdywt.35 g-45g). FLINT and FLINT analog (R218Q) were diluted in phosphatebuffered saline/10% glycerol to give dose solutions having a finalconcentration of 75 μg/ml. FLINT and FLINT analog (R21SQ) wereadministered to ICR mice (n=2) as a single intravenous bolus via thetail vein (15 μg/animal) in a volume of 0.2 ml. At 0.25 h afterintravenous administration of test articles, blood samples werecollected by cardiac puncture into EDTA tubes containing 0.01 ml of aprotease inhibitor cocktail (0.5 mM AEBSF, 150 rM aprotinin, 1 μm E-64,1 μM leupeptin) and plasma prepared by sedimentation. Plasmaconcentrations of FLINT or FLINT analog (R218Q) were determined by ELISAand the metabolic profile of FLINT immuoreactivity was determined usingreversed-phase HPLC as described below.

FLINT ELISA: Wells of a 96-well microtiter plate were coated overnightwith purified polyclonal antibody TKD-028-1494 (5 μg/ml, 0.1 ml/well).After washing, samples were added in a volume of 50 μl/well andincubated at room temperature for 2 h. After washing, biotinylated AbTKD-076A was added (1:4000 dilution, 50 μl/well) and incubated at roomtemperature for 1 h. After washing, streptavidin-alkaline phosphatase(Boehringer Ingleheim) was added (1:1000 dilution, 50 μl/well) andincubated at room temperature for 1 h. Detection was accomplished withAttophos™ substrate, 50 μl/well. Fluorescence intensity was measured at15 minute intervals at room temperature in a Biolumin. The LOQ of theassay was determined to be approximately 0.5 ng/ml.

The ELISA analysis showed that plasma concentrations of FLINT and PLINTanalog (R218Q) were comparable (approximately 200-300 ng/ml) whenmeasured 15 minutes after intravenous administration. However, in orderto distinguish between full length FLINT and metabolic FLINT (i.e.residues 1-218 of SEQ ID NO:1) RP-HLPC was carried out. Collectedfractions were concentrated to dryness in a Speed-Vac (SavantInstruments), resuspended in PBS/0.1% BSA and assayed by ELISA.

RP-HPLC fractionation indicated that FLINT metabolite (1-218)represented approximately 50% of the circulating immunoreactivity 15minutes after intravenous administration of native FLINT (FIG. 6A). Incontrast, FLINT metabolite was not observed in the plasma from animalsadministered FLINT analog (R218Q). Essentially all immunoreactivity wasaccounted for solely by the FLINT analog(R218Q), indicating resistanceto proteolytic degradation at the R218Q position (FIG. 6B).

EXAMPLE 15 Use of FLINT Analog to Treat ALI Patient

A 55 year-old male presents to the emergency department unconscious. Hisfamily states that he was being treated as an outpatient for bronchitisfor the past few days but worsened despite antibiotics. He has norelevant past history and his only medication was a third generationoral cephalosporin. Physical examination reveals an obtunded, cyanoticmale who is hypotensive, tachypneic, and tachycardic, and who hasbilateral lung congestion consistent with pulmonary edema. There is noevidence of congestive heart failure. Tests reveal hypoxemia (based onPaO2/FiO2) and bilateral lung infiltrates without cardiomegaly,consistent with a diagnosis of acute lung injury. Based on the historyit is concluded that the lung injury was a direct result ofcommunity-acquired pneumonia, and that the patient met the hypoxemiacriteria for ALI within the last 12 hours. Ventilation measures includeuse of PEEP and low tidal volume. As soon as adequate oxygenation isconfirmed, treatment with FLINT analog R218Q is initiated in the ER asan iv bolus of 2.5 mg/kg, followed by a continuous infusion of 0.1mg/minute. FLINT analog along with aggressive supportive measures (e.g.,positive ventilation, intravenous fluids, pressors, and nutritionalsupport) are continued for four days in the ICU, at which time the FLINTanalog is discontinued. Over the following 3 days, the patient begins torecover and is extubated on Day 8. He has an uneventful recovery and 6months following discharge has no evidence of residual lung disease byblood gas and spirometry.

EQUIVALENTS

Those skilled in the art will be able to recognize, or be able toascertain, using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

9 1 271 PRT Homo sapiens 1 Val Ala Glu Thr Pro Thr Tyr Pro Trp Arg AspAla Glu Thr Gly Glu 1 5 10 15 Arg Leu Val Cys Ala Gln Cys Pro Pro GlyThr Phe Val Gln Arg Pro 20 25 30 Cys Arg Arg Asp Ser Pro Thr Thr Cys GlyPro Cys Pro Pro Arg His 35 40 45 Tyr Thr Gln Phe Trp Asn Tyr Leu Glu ArgCys Arg Tyr Cys Asn Val 50 55 60 Leu Cys Gly Glu Arg Glu Glu Glu Ala ArgAla Cys His Ala Thr His 65 70 75 80 Asn Arg Ala Cys Arg Cys Arg Thr GlyPhe Phe Ala His Ala Gly Phe 85 90 95 Cys Leu Glu His Ala Ser Cys Pro ProGly Ala Gly Val Ile Ala Pro 100 105 110 Gly Thr Pro Ser Gln Asn Thr GlnCys Gln Pro Cys Pro Pro Gly Thr 115 120 125 Phe Ser Ala Ser Ser Ser SerSer Glu Gln Cys Gln Pro His Arg Asn 130 135 140 Cys Thr Ala Leu Gly LeuAla Leu Asn Val Pro Gly Ser Ser Ser His 145 150 155 160 Asp Thr Leu CysThr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val 165 170 175 Pro Gly AlaGlu Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe 180 185 190 Gln AspIle Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu 195 200 205 AlaPro Glu Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu 210 215 220Gln Leu Lys Leu Arg Arg Arg Leu Thr Glu Leu Leu Gly Ala Gln Asp 225 230235 240 Gly Ala Leu Leu Val Arg Leu Leu Gln Ala Leu Arg Val Ala Arg Met245 250 255 Pro Gly Leu Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val His260 265 270 2 813 DNA Homo sapiens 2 gtggcagaaa cacccaccta cccctggcgggacgcagaga caggggagcg gctggtgtgc 60 gcccagtgcc ccccaggcac ctttgtgcagcggccgtgcc gccgagacag ccccacgacg 120 tgtggcccgt gtccaccgcg ccactacacgcagttctgga actacctgga gcgctgccgc 180 tactgcaacg tcctctgcgg ggagcgtgaggaggaggcac gggcttgcca cgccacccac 240 aaccgtgcct gccgctgccg caccggcttcttcgcgcacg ctggtttctg cttggagcac 300 gcatcgtgtc cacctggtgc cggcgtgattgccccgggca cccccagcca gaacacgcag 360 tgccagccgt gccccccagg caccttctcagccagcagct ccagctcaga gcagtgccag 420 ccccaccgca actgcacggc cctgggcctggccctcaatg tgccaggctc ttcctcccat 480 gacaccctgt gcaccagctg cactggcttccccctcagca ccagggtacc aggagctgag 540 gagtgtgagc gtgccgtcat cgactttgtggctttccagg acatctccat caagaggctg 600 cagcggctgc tgcaggccct cgaggccccggagggctggg gtccgacacc aagggcgggc 660 cgcgcggcct tgcagctgaa gctgcgtcggcggctcacgg agctcctggg ggcgcaggac 720 ggggcgctgc tggtgcggct gctgcaggcgctgcgcgtgg ccaggatgcc cgggctggag 780 cggagcgtcc gtgagcgctt cctccctgtgcac 813 3 300 PRT Homo sapiens 3 Met Arg Ala Leu Glu Gly Pro Gly Leu SerLeu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro Val ProAla Val Arg Gly Val Ala Glu 20 25 30 Thr Pro Thr Tyr Pro Trp Arg Asp AlaGlu Thr Gly Glu Arg Leu Val 35 40 45 Cys Ala Gln Cys Pro Pro Gly Thr PheVal Gln Arg Pro Cys Arg Arg 50 55 60 Asp Ser Pro Thr Thr Cys Gly Pro CysPro Pro Arg His Tyr Thr Gln 65 70 75 80 Phe Trp Asn Tyr Leu Glu Arg CysArg Tyr Cys Asn Val Leu Cys Gly 85 90 95 Glu Arg Glu Glu Glu Ala Arg AlaCys His Ala Thr His Asn Arg Ala 100 105 110 Cys Arg Cys Arg Thr Gly PhePhe Ala His Ala Gly Phe Cys Leu Glu 115 120 125 His Ala Ser Cys Pro ProGly Ala Gly Val Ile Ala Pro Gly Thr Pro 130 135 140 Ser Gln Asn Thr GlnCys Gln Pro Cys Pro Pro Gly Thr Phe Ser Ala 145 150 155 160 Ser Ser SerSer Ser Glu Gln Cys Gln Pro His Arg Asn Cys Thr Ala 165 170 175 Leu GlyLeu Ala Leu Asn Val Pro Gly Ser Ser Ser His Asp Thr Leu 180 185 190 CysThr Ser Cys Thr Gly Phe Pro Leu Ser Thr Arg Val Pro Gly Ala 195 200 205Glu Glu Cys Glu Arg Ala Val Ile Asp Phe Val Ala Phe Gln Asp Ile 210 215220 Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu Ala Pro Glu 225230 235 240 Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu Gln LeuLys 245 250 255 Leu Arg Arg Arg Leu Thr Glu Leu Leu Gly Ala Gln Asp GlyAla Leu 260 265 270 Leu Val Arg Leu Leu Gln Ala Leu Arg Val Ala Arg MetPro Gly Leu 275 280 285 Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val His290 295 300 4 29 PRT Homo sapiens 4 Met Arg Ala Leu Glu Gly Pro Gly LeuSer Leu Leu Cys Leu Val Leu 1 5 10 15 Ala Leu Pro Ala Leu Leu Pro ValPro Ala Val Arg Gly 20 25 5 39 DNA Artificial Sequence Description ofArtificial Sequence oligo primer 5 gcaccagggt accaggagct gaggagtgtgagcgtgccg 39 6 44 DNA Artificial Sequence Description of ArtificialSequence oligo primer 6 tcagctgcaa ggcggcgcgc cccgcttgtg gtgtcggacc ccag44 7 44 DNA Artificial Sequence Description of Artificial Sequence oligoprimer 7 ggggtccgac accacaagcg gggcgcgccg ccttgcagct gaag 44 8 43 DNAArtificial Sequence Description of Artificial Sequence oligo primer 8gcacagaatt catcagtgca cagggaggaa gcgctcacgg acg 43 9 936 DNA Homosapiens CDS (25)..(924) 9 gctctccctg ctccagcaag gacc atg agg gcg ctg gagggg cca ggc atg 53 Met Arg Ala Leu Glu Gly Pro Gly Leu 1 5 tcg ctg ctgtgc ctg gtg ttg gcg ctg cct gcc ctg ctg ccg gtg ccg 99 Ser Leu Leu CysLeu Val Leu Ala Leu Pro Ala Leu Leu Pro Val Pro 10 15 20 25 gct gta cgcgga gtg gca gaa aca ccc acc tac ccc tgg cgg gac gca 147 Ala Val Arg GlyVal Ala Glu Thr Pro Thr Tyr Pro Trp Arg Asp Ala 30 35 40 gag aca ggg gagcgg ctg gtg tgc gcc cag tgc ccc cca ggc acc ttt 195 Glu Thr Gly Glu ArgLeu Val Cys Ala Gln Cys Pro Pro Gly Thr Phe 45 50 55 gtg cag cgg ccg tgccgc cga gac agc ccc acg acg tgt ggc ccg tgt 243 Val Gln Arg Pro Cys ArgArg Asp Ser Pro Thr Thr Cys Gly Pro Cys 60 65 70 cca ccg cgc cac tac acgcag ttc tgg aac tac ctg gag cgc tgc cgc 291 Pro Pro Arg His Tyr Thr GlnPhe Trp Asn Tyr Leu Glu Arg Cys Arg 75 80 85 tac tgc aac gtc ctc tgc ggggag cgt gag gag gag gca cgg gct tgc 339 Tyr Cys Asn Val Leu Cys Gly GluArg Glu Glu Glu Ala Arg Ala Cys 90 95 100 105 cac gcc acc cac aac cgtgcc tgc cgc tgc cgc acc ggc ttc ttc gcg 387 His Ala Thr His Asn Arg AlaCys Arg Cys Arg Thr Gly Phe Phe Ala 110 115 120 cac gct ggt ttc tgc ttggag cac gca tcg tgt cca cct ggt gcc ggc 435 His Ala Gly Phe Cys Leu GluHis Ala Ser Cys Pro Pro Gly Ala Gly 125 130 135 gtg att gcc ccg ggc accccc agc cag aac acg cag tgc cag ccg tgc 483 Val Ile Ala Pro Gly Thr ProSer Gln Asn Thr Gln Cys Gln Pro Cys 140 145 150 ccc cca ggc acc ttc tcagcc agc agc tcc agc tca gag cag tgc cag 531 Pro Pro Gly Thr Phe Ser AlaSer Ser Ser Ser Ser Glu Gln Cys Gln 155 160 165 ccc cac cgc aac tgc acggcc ctg ggc ctg gcc ctc att gtg cca ggc 579 Pro His Arg Asn Cys Thr AlaLeu Gly Leu Ala Leu Ile Val Pro Gly 170 175 180 185 tct tcc tcc cat gacacc ctg tgc acc agc tgc act ggc ttc ccc ctc 627 Ser Ser Ser His Asp ThrLeu Cys Thr Ser Cys Thr Gly Phe Pro Leu 190 195 200 agc acc agg gta ccagga gct gag gag tgt gag cgt gcc gtc atc gac 675 Ser Thr Arg Val Pro GlyAla Glu Glu Cys Glu Arg Ala Val Ile Asp 205 210 215 ttt gtg gct ttc caggac atc tcc atc aag agg ctg cag cgg ctg ctg 723 Phe Val Ala Phe Gln AspIle Ser Ile Lys Arg Leu Gln Arg Leu Leu 220 225 230 cag gcc ctc gag gccccg gag ggc tgg gct ccg aca cca agg gcg ggc 771 Gln Ala Leu Glu Ala ProGlu Gly Trp Ala Pro Thr Pro Arg Ala Gly 235 240 245 cgc gcg gcc ttg cagctg aag ctg cgt cgg cgg ctc acg gag ctc ctg 819 Arg Ala Ala Leu Gln LeuLys Leu Arg Arg Arg Leu Thr Glu Leu Leu 250 255 260 265 ggg gcg cag gacggg gcg ctg ctg gtg cgg ctg ctg cag gcg ctg cgc 867 Gly Ala Gln Asp GlyAla Leu Leu Val Arg Leu Leu Gln Ala Leu Arg 270 275 280 gtg gcc agg atgccc ggg ctg gag cgg agc gtc cgt gag cgc ttc ctc 915 Val Ala Arg Met ProGly Leu Glu Arg Ser Val Arg Glu Arg Phe Leu 285 290 295 cct gtg cactgatcctggc cc 936 Pro Val His 300

What is claimed is:
 1. A FLINT analog resistant to proteolysis atposition 218 of SEO ID NO:1 comprising the priority of SEQ ID NO:1, withthe exception that the residue of position 218 is a substitutionselected from the group consisting of: a. Arg at position 218 isreplaced by Gln b. Arg at position 218 is replaced by Glu; c. Arg atposition 218 is replaced by Ala; d. Arg at position 218 is replaced byGly; e. Arg at position 218 is replaced by Ser; f. Arg at position 218is replaced by Val; g. Arg at position 218 is replaced by Tyr, and h.Arg at position 218 is replaced by Asn.
 2. A FLINT analog wherein Thr atposition 216 of SEQ ID NO:1 is replaced by Pro, and Arg at position 218is replaced by Gln.
 3. A method to treat or prevent a disease orcondition in a mammal comprising the administration of a therapeuticallyeffective amount of a protease resistant FLINT analog of claim 1 orclaim
 2. 4. A method as in claim 3 wherein said disease or condition isacute lung injury, acute respiratory distress syndrome, or ulcerativecolitis.
 5. A pharmaceutical formulation comprising as an activeingredient a protease resistant FLINT analog of claim 1 or claim 2associated with one or more pharmaceutically acceptable carriers,excipients, or diluents thereof.
 6. A FLINT analog resistant toproteolysis at position 218 of SEQ ID NO:1 comprising the amino acidsequence of SEQ ID NO:1, wherein Arg at position 218 is substituted byGln.